Pyruvate kinase activators for use in treating blood disorders

ABSTRACT

Described herein are compounds that activate pyruvate kinase, pharmaceutical compositions and methods of use thereof. These compounds are represented by Formula (I) wherein R 1 , R 2 , R a , R b , R j , R k , and Q are as defined herein

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority of U.S. ProvisionalPatent Application Nos. 62/673,526 and 62/673,533, both filed May 18,2018. This application also claims the benefit of priority ofInternational Patent Application No. PCT/CN2017/097496, filed Aug. 15,2017. Each of the aforementioned priority applications is incorporatedherein by reference in its entirety.

BACKGROUND

Pyruvate kinase deficiency (PKD) is a disease of the red blood cellscaused by a deficiency of the pyruvate kinase R (PKR) enzyme due torecessive mutations of PKLR gene (Wijk et al. Human Mutation, 2008, 30(3) 446-453). PKR activators can be beneficial to treat PKD, thalassemia(e.g., beta-thalessemia), hereditary elliptocytosis,abetalipoproteinemia or Bassen-Kornzweig syndrome, sickle cell disease,paroxysmal nocturnal hemoglobinuria, anemia (e.g., congenital anemias(e.g., enzymopathies), hemolytic anemia (e.g. hereditary and/orcongenital hemolytic anemia, acquired hemolytic anemia, chronichemolytic anemia caused by phosphoglycerate kinase deficiency, anemia ofchronic diseases, non-spherocytic hemolytic anemia or hereditaryspherocytosis).

SUMMARY

Described herein are compounds of Formulas (I), (II), (III), (IV), and(V) (collectively referred to herein as “Formulas (I)-(V)”), thatactivate pyruvate kinase R (PKR), wild type and/or mutant enzymes (suchas those described herein), and pharmaceutically acceptable saltsthereof.

In one embodiment, the invention provides a compound of Formula (I) or apharmaceutically acceptable salt thereof:

wherein R¹, R², R^(a), R^(b), R^(j), R^(k), and Q are as defined herein.

In one embodiment, the compound or pharmaceutically acceptable saltthereof is selected from the compounds of Table 1 or FIG. 1.

Also provided are pharmaceutical compositions comprising a compound ofFormulas (I)-(V), or a pharmaceutically acceptable salt thereof, and apharmaceutically acceptable carrier.

The present disclosure further provides a method of treating anemia in asubject comprising administering to the subject an effective amount of(1) a compound described herein or a pharmaceutically acceptable saltthereof; (2) a pharmaceutically acceptable composition comprising acompound described herein or a pharmaceutically acceptable salt thereof,and a pharmaceutically acceptable carrier. In certain embodiments, theanemia is a dyserythropoietic anemia such as congenitaldyserythropoietic anemia type I, II, III, or IV.

The present disclosure further provides a method for treating sicklecell disease in a subject comprising administering to the subject aneffective amount of (1) a compound disclosed herein or apharmaceutically acceptable salt thereof; (2) a pharmaceuticalcomposition comprising a compound disclosed herein or a pharmaceuticallyacceptable salt thereof, and a pharmaceutically acceptable carrier.

The present disclosure further provides a method for treating hemolyticanemia (e.g., chronic hemolytic anemia caused by phosphoglycerate kinasedeficiency, Blood Cells Mol Dis, 2011; 46(3):206) in a subjectcomprising administering to the subject an effective amount of (1) acompound disclosed herein or a pharmaceutically acceptable salt thereof;(2) a pharmaceutical composition comprising a compound disclosed hereinor a pharmaceutically acceptable salt thereof, and a pharmaceuticallyacceptable carrier. In certain embodiments, the hemolytic anemia ishereditary and/or congenital hemolytic anemia, acquired hemolyticanemia, or anemia as part of a multi-system disease. In certainembodiments, the hemolytic anemia is congenital anemia. In certainembodiments, the hemolytic anemia is hereditary (e.g. non-spherocytichemolytic anemia or hereditary spherocytosis).

The present disclosure further provides a method for treatingthalassemia (e.g., beta-thalassemia), hereditary spherocytosis,hereditary elliptocytosis, abetalipoproteinemia (or Bassen-Kornzweigsyndrome), paroxysmal nocturnal hemoglobinuria, acquired hemolyticanemia (e.g., congenital anemias (e.g., enzymopathies)), sickle celldisease, or anemia of chronic diseases in a subject comprisingadministering to the subject an effective amount of (1) a compounddisclosed herein or a pharmaceutically acceptable salt thereof; (2) apharmaceutical composition comprising a compound disclosed herein or apharmaceutically acceptable salt thereof, and a pharmaceuticallyacceptable carrier. In one embodiment, the acquired hemolytic anemiacomprises congenital anemias. In certain embodiments, the providedmethod is to treat thalassemia. In certain embodiments, the thalassemiais beta-thalassemia.

The present disclosure further provides a method for treating pyruvatekinase deficiency (PKD) in a subject, the method comprisingadministering to the subject an effective amount of (1) a compounddisclosed herein or a pharmaceutically acceptable salt thereof; (2) apharmaceutical composition comprising a compound disclosed herein or apharmaceutically acceptable salt thereof, and a pharmaceuticallyacceptable carrier. In certain embodiments, the PKD is a deficiency ofPKR. In certain embodiments, the deficiency of PKR is associated with apyruvate kinase R mutation.

Compounds and pharmaceutical compositions described herein areactivators of PKR having lower activities compared to the wild type,thus are useful for methods of the present disclosure. In certainembodiments, the PKR is a wild type. In certain embodiments, the PKR isa mutant. Such mutations in PKR can affect enzyme activity (catalyticefficiency), regulatory properties (modulation by fructose bisphosphate(FBP)/ATP), and/or thermostability of the enzyme. Examples of suchmutations are described in Valentini et al, JBC 2002. Some examples ofthe mutants that are activated by the compounds described herein includeG332S, G364D, T384M, R479H, R479K, R486W, R532W, K410E, R510Q, andR490W. Without being bound by theory, in certain embodiments, thecompounds described herein affect the activities of PKR mutants byactivating FBP non-responsive PKR mutants, restoring thermostability tomutants with decreased stability, or restoring catalytic efficiency toimpaired mutants. The activating activity of the present compoundsagainst PKR mutants may be tested following a method described in theExamples. In certain embodiments, the compounds described herein arealso activators of wild type PKR.

In an embodiment, the disclosure provides a method for activating PKR inred blood cells in a subject in need thereof comprising administering tothe subject an effective amount of (1) a compound disclosed herein or apharmaceutically acceptable salt thereof; (2) a pharmaceuticalcomposition comprising a compound disclosed herein or a pharmaceuticallyacceptable salt thereof, and a pharmaceutically acceptable carrier. Incertain embodiments, the PKR is a wild type. In certain embodiments, thePKR is a mutant.

In an embodiment, the mutant PKR is selected from G332S, G364D, T384M,K410E, R479H, R479K, R486W, R532W, R510Q, and R490W. In certainembodiments, the mutant PKR is selected from A468V, A495V, I90N, T408I,and Q421K, and R498H. In certain embodiments, the mutant PKR is R532W,K410E, or R510Q.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a listing of the structures of exemplary compounds of theinvention.

FIG. 2 shows synthesis of exemplary intermediates used in Examples 1-10.

DETAILED DESCRIPTION OF THE INVENTION

The details of construction and the arrangement of components set forthin the following description or illustrated in the drawings are notmeant to be limiting. Embodiments can be practiced or carried out invarious ways. The phraseology and terminology used herein is for purposeof description and shouldn't be regarded as limiting.

Definitions

Compounds described herein can comprise one or more asymmetric centers,and thus can exist in various stereoisomeric forms, e.g., enantiomersand/or diastereomers. For example, the compounds described herein can bein the form of an individual enantiomer, diastereomer or geometricisomer, or can be in the form of a mixture of stereoisomers, includingracemic mixtures and mixtures enriched in one or more stereoisomer.Isomers can be isolated from mixtures by methods known to those skilledin the art, including chiral high pressure liquid chromatography (HPLC)and the formation and crystallization of chiral salts; or preferredisomers can be prepared by asymmetric syntheses. See, for example,Jacques et al., Enantiomers, Racemates and Resolutions (WileyInterscience, New York, 1981); Wilen et al., Tetrahedron 33:2725 (1977);Eliel, E. L. Stereochemistry of Carbon Compounds (McGraw-Hill, N Y,1962); and Wilen, S. H. Tables of Resolving Agents and OpticalResolutions p. 268 (E. L. Eliel, Ed., Univ. of Notre Dame Press, NotreDame, Ind. 1972).

The compounds described herein may also be represented in multipletautomeric forms, in such instances, expressly includes all tautomericforms of the compounds described herein, even though only a singletautomeric form may be represented (e.g., alkylation of a ring systemmay result in alkylation at multiple sites; all such reaction productsare expressly included). All such isomeric forms of such compounds areexpressly included. If a tautomer of a compound is aromatic, thiscompound is aromatic. Similarly, if a tautomer of a substitutent is aheteroaryl, this substituent is heteroaryl.

The term “alkyl” refers to a radical of a straight-chain or branchedsaturated hydrocarbon group having from 1 to 10 carbon atoms (“C₁₋₁₀alkyl”). Examples of C₁₋₆ alkyl groups include methyl (C₁), ethyl (C₂),propyl (C₃) (e.g., n-propyl, isopropyl), butyl (C₄) (e.g., n-butyl,tert-butyl, sec-butyl, iso-butyl), pentyl (C₅) (e.g., n-pentyl,3-pentanyl, amyl, neopentyl, 3-methyl-2-butanyl, tertiary amyl), andhexyl (C₆) (e.g., n-hexyl). Unless otherwise specified, each instance ofan alkyl group is independently unsubstituted (an “unsubstituted alkyl”)or substituted (a “substituted alkyl”) with one or more substituents(e.g., halogen, such as F). In certain embodiments, the alkyl group isunsubstituted —C₁₋₁₀ alkyl. In certain embodiments, the alkyl group issubstituted —C₁₋₁₀ alkyl.

The term “haloalkyl” refers to a substituted alkyl group, wherein one ormore of the hydrogen atoms are independently replaced by a halogen,e.g., fluoro, bromo, chloro, or iodo and includes alkyl moieties inwhich all hydrogens have been replaced by halo (e.g., perfluoroalkyl).In some embodiments, the haloalkyl moiety has 1 to 8 carbon atoms (“C₁₋₈haloalkyl”).

The term “alkoxy” or “alkoxyl” refers to an —O-alkyl radical. E.g., withbetween 1 and 6 carbon atoms.

The term “aryloxy” refers to an —O-aryl radical. In some embodiments thearyloxy group is phenoxy.

The term “alkenyl” refers to a radical of a straight-chain or branchedhydrocarbon group having from 2 to 10 carbon atoms and one or morecarbon-carbon double bonds (e.g., 1, 2, 3, or 4 double bonds). The oneor more carbon-carbon double bonds can be internal (such as in2-butenyl) or terminal (such as in 1-butenyl). Examples of —C₂₋₄ alkenylgroups include ethenyl (C₂), 1-propenyl (C₃), 2-propenyl (C₃), 1-butenyl(C₄), 2-butenyl (C₄), butadienyl (C₄), pentenyl (C₅), pentadienyl (C₅),hexenyl (C₆), heptenyl (C₇), octenyl (C₈), octatrienyl (C₈), and thelike. Unless otherwise specified, each instance of an alkenyl group isindependently unsubstituted (an “unsubstituted alkenyl”) or substituted(a “substituted alkenyl”) with one or more substituents. In certainembodiments, the alkenyl group is an unsubstituted —C₂₋₁₀ alkenyl. Incertain embodiments, the alkenyl group is a substituted —C₂₋₁₀ alkenyl.In an alkenyl group, a C═C double bond may be an (E)- or (Z)-doublebond.

The term “alkynyl” refers to a radical of a straight-chain or branchedhydrocarbon group having from 2 to 10 carbon atoms and one or morecarbon-carbon triple bonds (e.g., 1, 2, 3, or 4 triple bonds) (“C₂₋₁₀alkynyl”). Examples of alkynyl groups include ethynyl (C₂), 1-propynyl(C₃), 2-propynyl (C₃), 1-butynyl (C₄), 2-butynyl (C₄), pentynyl (C₅),hexynyl (C₆) heptynyl (C₇), octynyl (C₈), and the like. Unless otherwisespecified, each instance of an alkynyl group is independentlyunsubstituted (an “unsubstituted alkynyl”) or substituted (a“substituted alkynyl”) with one or more substituents. In certainembodiments, the alkynyl group is an unsubstituted —C₂₋₁₀ alkynyl. Incertain embodiments, the alkynyl group is a substituted —C₂₋₁₀ alkynyl.

The term “carbocyclyl” or “carbocyclic” refers to a radical of anon-aromatic monocyclic, bicyclic, or tricyclic or polycyclichydrocarbon ring system having from 3 to 14 ring carbon atoms (“C₃₋₁₄carbocyclyl”) and zero heteroatoms in the non-aromatic ring system.Carbocyclyl groups include fully saturated ring systems (e.g.,cycloalkyls), and partially saturated ring systems. In some embodiments,a carbocyclyl group has 3 to 10 ring carbon atoms (“C₃₋₁₀ carbocyclyl”).

The term “cycloalkyl” as employed herein includes saturated cyclic,bicyclic, tricyclic, or polycyclic hydrocarbon groups having 3 to 14carbons containing the indicated number of rings and carbon atoms (forexample a C₃-C₁₄ monocyclic, C₄-C₁₄ bicyclic, C₅-C₁₄ tricyclic, orC₆-C₁₄ polycyclic cycloalkyl). In some embodiments “cycloalkyl” is amonocyclic cycloalkyl. Examples of monocyclic cycloalkyl groups includecyclopentyl (C₅), cyclohexyl (C₅), cyclopropyl (C₃) cyclobutyl (C₄),cycloheptyl (C₇) and cyclooctyl (C₈). In some embodiments “cycloalkyl”is a bicyclic cycloalkyl. Examples of bicyclic cycloalkyls includebicyclo[1.1.0]butane (C₄), bicyclo[1.1.1]pentane (C₅), spiro[2.2]pentane(C₅), bicyclo[2.1.0]pentane (C₅), bicyclo[2.1.1]hexane (C₆),bicyclo[3.3.3]undecane (C₁₁), decahydronaphthalene (C₁₀),bicyclo[4.3.2]undecane (C₁₁), spiro[5.5]undecane (C₁₁) andbicyclo[4.3.3]dodecane (C₁₂). In some embodiments “cycloalkyl” is atricyclic cycloalkyl. Examples of tricyclic cycloalkyls includeadamantine (C₁₂). Unless otherwise specified, each instance of acycloalkyl group is independently unsubstituted (an “unsubstitutedcycloalkyl”) or substituted (a “substituted cycloalkyl”) with one ormore substituents. In certain embodiments, the cycloalkyl group is anunsubstituted C₃₋₁₄ cycloalkyl. In certain embodiments, the cycloalkylgroup is a substituted C₃₋₁₄ cycloalkyl.

The term “heterocyclyl” or “heterocylic” refers to a radical of a 3- to14-membered non-aromatic ring system having ring carbon atoms and 1 to 4ring heteroatoms, wherein each heteroatom is independently selected fromnitrogen, oxygen, and sulfur (“3-14 membered heterocyclyl”). Inheterocyclyl groups that contain one or more nitrogen atoms, the pointof attachment can be a carbon or nitrogen atom, as valency permits. Aheterocyclyl group can either be monocyclic (“monocyclic heterocyclyl”)or polycyclic (e.g., a fused, bridged or spiro ring system such as abicyclic system (“bicyclic heterocyclyl”) or tricyclic system(“tricyclic heterocyclyl”)), and can be saturated or can contain one ormore carbon-carbon double or triple bonds. Heterocyclyl polycyclic ringsystems can include one or more heteroatoms in one or both rings.“Heterocyclyl” also includes ring systems wherein the heterocyclyl ring,as defined above, is fused with one or more carbocyclyl groups whereinthe point of attachment is either on the carbocyclyl or heterocyclylring, or ring systems wherein the heterocyclyl ring, as defined above,is fused with one or more aryl or heteroaryl groups, wherein the pointof attachment is on the heterocyclyl ring, and in such instances, thenumber of ring members continue to designate the number of ring membersin the heterocyclyl ring system. Unless otherwise specified, eachinstance of heterocyclyl is independently unsubstituted (an“unsubstituted heterocyclyl”) or substituted (a “substitutedheterocyclyl”) with one or more substituents. In certain embodiments,the heterocyclyl group is an unsubstituted 3-14 membered heterocyclyl.In certain embodiments, the heterocyclyl group is a substituted 3-14membered heterocyclyl. In some embodiments, a heterocyclyl group is a5-10 membered non-aromatic ring system having ring carbon atoms and 1-4ring heteroatoms, wherein each heteroatom is independently selected fromnitrogen, oxygen, and sulfur (“5-10 membered heterocyclyl”). Exemplaryheterocyclyl groups include aziridinyl, oxiranyl, thiiranyl, azetidinyl,oxetanyl, thietanyl, tetrahydrofuranyl, dihydrofuranyl,tetrahydrothiophenyl, dihydrothiophenyl, pyrrolidinyl, dihydropyrrolyl,pyrrolyl-2,5-dine, dioxolanyl, oxathiolanyl, dithiolanyl, triazolinyl,oxadiazolinyl, thiadiazolinyl, piperidinyl, tetrahydropyranyl,dihydropyridinyl, thianyl, piperazinyl, morpholinyl, dithianyl,dioxanyl, triazinanyl, azepanyl, oxepanyl, thiepanyl, azocanyl,oxecanyl, thiocanyl, indolinyl, isoindolinyl, dihydrobenzofuranyl,dihydrobenzothienyl, tetrahydrobenzothienyl, tetrahydrobenzofuranyl,tetrahydroindolyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl,decahydroquinolinyl, decahydroisoquinolinyl, octahydrochromenyl,octahydroisochromenyl, decahydronaphthyridinyl,decahydro-1,8-naphthyridinyl, octahydropyrrolo[3,2-b]pyrrole, indolinyl,phthalimidyl, naphthalimidyl, chromanyl, chromenyl,1H-benzo[e][1,4]diazepinyl, 1,4,5,7-tetrahydropyrano[3,4-b]pyrrolyl,5,6-dihydro-4H-furo[3,2-b]pyrrolyl, 6,7-dihydro-5H-furo[3,2-b]pyranyl,5,7-dihydro-4H-thieno[2,3-c]pyranyl,2,3-dihydro-1H-pyrrolo[2,3-b]pyridinyl, 2,3-dihydrofuro[2,3-b]pyridinyl,4,5,6,7-tetrahydro-1H-pyrrolo[2,3-b]pyridinyl,4,5,6,7-tetrahydrofuro[3,2-c]pyridinyl,4,5,6,7-tetrahydrothieno[3,2-b]pyridinyl,1,2,3,4-tetrahydro-1,6-naphthyridinyl, and the like.

The term “aryl” refers to a radical of a monocyclic or polycyclic (e.g.,bicyclic or tricyclic) 4n+2 aromatic ring system (e.g., having 6, 10, or14× electrons shared in a cyclic array) having 6-14 ring carbon atomsand zero heteroatoms provided in the aromatic ring system (“C₆₋₁₄aryl”). In some embodiments, an aryl group has 6 ring carbon atoms (“C₆aryl”; e.g., phenyl). In some embodiments, an aryl group has 10 ringcarbon atoms (“C₁₀ aryl”; e.g., naphthyl such as 1-naphthyl and2-naphthyl). In some embodiments, an aryl group has 14 ring carbon atoms(“C₁₋₄ aryl”; e.g., anthracyl). “Aryl” also includes ring systemswherein the aryl ring, as defined above, is fused with one or morecarbocyclyl or heterocyclyl groups wherein the radical or point ofattachment is on the aryl ring, and in such instances, the number ofcarbon atoms continue to designate the number of carbon atoms in thearyl ring system. Unless otherwise specified, each instance of an arylgroup is independently unsubstituted (an “unsubstituted aryl”) orsubstituted (a “substituted aryl”) with one or more substituents. Incertain embodiments, the aryl group is an unsubstituted C₆₋₁₄ aryl. Incertain embodiments, the aryl group is a substituted C₆₋₁₄ aryl.

The term “heteroaryl” refers to a radical of a 5-14 membered monocyclicor polycyclic (e.g., bicyclic, tricyclic) 4n+2 aromatic ring system(e.g., having 6× electrons shared in a cyclic array) having ring carbonatoms and 1-4 ring heteroatoms provided in the aromatic ring system,wherein each heteroatom is independently selected from nitrogen, oxygen,and sulfur (“5-14 membered heteroaryl”). In some embodiments, theheteroaryl can be a 5- or 6-membered monocyclic heteroaryl containing1-4 heteroatoms. In some embodiments the heteroaryl can be an 8-12membered bicyclic heteroaryl having 1-6 heteroatoms. A “5- or 6-memberedmonocyclic heteroaryl” or “5-membered or 6-membered monocyclicheteroaryl” refers to a 5- or 6-membered monocyclic and unfused 4n+2aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms.Exemplary monocyclic 5- or 6-membered heteroaryl groups includepyrrolyl, furanyl, thiophenyl, imidazolyl, pyrazolyl, oxazolyl,isoxazolyl, thiazolyl, isothiazolyl, triazolyl, oxadiazolyl,thiadiazolyl, tetrazolyl, pyridinyl, pyridazinyl, pyrimidinyl,pyrazinyl, triazinyl, and tetrazinyl. As used herein, if a tautomer of aradical is heteroaryl, this radical is heteroaryl. The term “tautomers”or “tautomeric” refers to two or more interconvertiblecompounds/substituents resulting from at least one formal migration of ahydrogen atom and at least one change in valency (e.g., a single bond toa double bond, a triple bond to a single bond, or vice versa). The exactratio of the tautomers depends on several factors, includingtemperature, solvent, and pH. Tautomerizations (i.e., the reactionproviding a tautomeric pair) may catalyzed by acid or base. Exemplarytautomerizations include keto-to-enol, amide-to-imide, lactam-to-lactim,enamine-to-imine, and enamine-to-(a different enamine) tautomerizations.

The term “saturated” refers to a moiety that does not contain a doubleor triple bond, i.e., the moiety only contains single bonds.

The term “optionally substituted” refers to being substituted orunsubstituted. In general, the term “substituted” means that at leastone hydrogen present on a group is replaced with a permissiblesubstituent, e.g., a substituent which upon substitution results in astable compound, e.g., a compound which does not spontaneously undergotransformation such as by rearrangement, cyclization, elimination, orother reaction. Unless otherwise indicated, a “substituted” group has asubstituent (e.g. C₄₋₆ alkyl, halogen, nitro, cyano, hydroxyl, C₁₋₆haloalkyl, C₁₋₆ haloalkoxy, C₁₋₆ acyl, C₃₋₆ cycloalkyl, C₆₋₁₀ aryl,monocyclic or bicyclic heteroaryl, and monocyclic or bicyclicheterocyclyl), at one or more substitutable positions of the group, andwhen more than one position in any given structure is substituted, thesubstituent is either the same or different at each position. The term“substituted” is contemplated to include substitution with allpermissible substituents of organic compounds, and includes any of thesubstituents described herein that results in the formation of a stablecompound. The present invention contemplates any and all suchcombinations in order to arrive at a stable compound. For purposes ofthis invention, heteroatoms such as nitrogen may have hydrogensubstituents and/or any suitable substituent as described herein whichsatisfy the valences of the heteroatoms and results in the formation ofa stable moiety. The invention is not intended to be limited in anymanner by the exemplary substituents described herein.

A “substitutable ring carbon atom” refers to a carbon atom on anaryl/heteroaryl/carbocyclyl/heterocyclyl ring with at least one hydrogenpresent on the carbon atom that is replaced with a permissiblesubstituent as defined above. A “substitutable ring nitrogen atom”refers to a nitrogen atom on an heteroaryl-heterocyclyl ring with atleast one hydrogen present on the nitrogen atom that is replaced with apermissible substituent.

Unless otherwise indicated, a “substituted” group has a substituent atone or more substitutable positions of the group, and when more than oneposition in any given structure is substituted, the substituent iseither the same or different at each position. The term “substituted” iscontemplated to include substitution with all permissible substituentsof organic compounds, and includes any of the substituents describedherein that results in the formation of a stable compound. The presentinvention contemplates any and all such combinations in order to arriveat a stable compound. For purposes of this invention, heteroatoms suchas nitrogen may have hydrogen substituents and/or any suitablesubstituent as described herein which satisfy the valences of theheteroatoms and results in the formation of a stable moiety. Theinvention is not intended to be limited in any manner by the exemplarysubstituents described herein.

The term “halo” or “halogen” refers to fluorine, chlorine, bromine, oriodine.

The term “acyl” refers to a group having the general formula—C(═O)R^(X1), —C(═O)OR^(X1), —C(═O)—O—C(═O)R^(X1), —C(═O)SR^(X1),—C(═O)N(R^(X1))₂, —C(═S)R^(X1), —C(═S)N(R^(X1))₂, and —C(═S)S(R^(X1)),—C(═NR^(X1))R^(X1), —C(═NR^(X1))OR^(X1), —C(═NR^(X1))SR^(X1), and—C(═NR^(X1))N(R^(X1))₂, wherein R^(X1) is hydrogen; halogen; substitutedor unsubstituted hydroxyl; substituted or unsubstituted thiol;substituted or unsubstituted amino; substituted or unsubstituted acyl,cyclic or acyclic, substituted or unsubstituted, branched or unbranchedC₁₋₁₀ alkyl; cyclic or acyclic, substituted or unsubstituted, branchedor unbranched C₂₋₁₀ alkenyl; substituted or unsubstituted C₂₋₁₀ alkynyl;substituted or unsubstituted C₆₋₁₂ aryl, substituted or unsubstitutedheteroaryl, when valency permits. Exemplary acyl groups includealdehydes (—CHO), carboxylic acids (—CO₂H), ketones, acyl halides,esters, amides, imines, carbonates, carbamates, and ureas.

In certain embodiments, the substituent present on a nitrogen atom, onan oxygen atom or on a sulfur atom is a nitrogen protecting group, anoxygen protecting group or a sulfur protecting group, respectively.Nitrogen, oxygen and sulfur protecting groups are well known in the artand include those described in detail in Protecting Groups in OrganicSynthesis, T. W. Greene and P. G. M. Wuts, 3^(rd) edition, John Wiley &Sons, 1999, incorporated herein by reference. For example, nitrogenprotecting groups include, but are not limited to, formamide, acetamide,chloroacetamide, trichloroacetamide, trifluoroacetamide,phenylacetamide, methyl carbamate, ethyl carbamate, 9-fluorenylmethylcarbamate (Fmoc), t-butyl carbamate (BOC or Boc), 1-adamantyl carbamate(Adoc), vinyl carbamate (Voc), allyl carbamate (Alloc),2-(trimethylsilyl) ethoxy]methyl (SEM), p-toluenesulfonamide (Ts),benzenesulfonamide, 2,3,6-trimethyl-4-methoxybenzenesulfonamide (Mtr),2,4,6-trimethoxybenzenesulfonamide (Mtb), phenothiazinyl-(10)-acylderivative, N′-p-toluenesulfonylaminoacyl derivative, etc. Exemplaryoxygen protecting groups include, but are not limited to, methyl,methoxymethyl (MOM), methylthiomethyl (MTM), t-butylthiomethyl,(phenyldimethylsilyl) methoxymethyl (SMOM), benzyloxymethyl (BOM),p-methoxybenzyloxymethyl (PMBM), (4-methoxyphenoxy)methyl (p-AOM),tetrahydropyranyl (THP), methanesulfonate (mesylate), benzylsulfonate,and tosylate (Ts).

The term “leaving group” is given its ordinary meaning in the art ofsynthetic organic chemistry and refers to an atom or a group capable ofbeing displaced by a nucleophile. Examples of suitable leaving groupsinclude, but are not limited to, halogen (such as F, Cl, Br, or I(iodine)), alkoxycarbonyloxy, aryloxycarbonyloxy, alkanesulfonyloxy,arenesulfonyloxy, alkyl-carbonyloxy (e.g., acetoxy), a sulfonic acidester, such as toluenesulfonate (tosylate, —OTs), methanesulfonate(mesylate, —OMs), p-bromobenzenesulfonyloxy (brosylate, —OBs),—OS(═O)₂(CF₂)₃CF₃ (nonaflate, —ONf), ortrifluoromethanesulfonate(triflate, —OTf).

The term “pharmaceutically acceptable salt” refers to those salts whichare, within the scope of sound medical judgment, suitable for use incontact with the tissues of humans and lower animals without unduetoxicity, irritation, allergic response, and the like, and arecommensurate with a reasonable benefit/risk ratio. Pharmaceuticallyacceptable salts are well known in the art. For example, Berge et al.describe pharmaceutically acceptable salts in detail in J.Pharmaceutical Sciences, 1977, 66, 1-19, incorporated herein byreference. Pharmaceutically acceptable salts of the compounds of thisinvention include those derived from suitable inorganic and organicacids and bases. Examples of pharmaceutically acceptable, nontoxic acidaddition salts are salts of an amino group formed with inorganic acids,such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuricacid, and perchloric acid or with organic acids, such as acetic acid,oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, ormalonic acid or by using other methods known in the art such as ionexchange. Other pharmaceutically acceptable salts include adipate,alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate,borate, butyrate, camphorate, camphorsulfonate, citrate,cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate,formate, fumarate, glucoheptonate, glycerophosphate, gluconate,hemisulfate, heptanoate, hexanoate, hydroiodide,2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, laurylsulfate, malate, maleate, malonate, methanesulfonate,2-naphthalenesulfonate, nicutinate, nitrate, oleate, oxalate, palmitate,pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate,pivalate, propionate, stearate, succinate, sulfate, tartrate,thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and thelike. Salts derived from appropriate bases include alkali metal,alkaline earth metal, ammonium, and N⁺(C₁₋₄ alkyl)₄ ⁻ salts.Representative alkali or alkaline earth metal salts include sodium,lithium, potassium, calcium, magnesium, and the like. Furtherpharmaceutically acceptable salts include, when appropriate, nontoxicammonium, quaternary ammonium, and amine cations formed usingcounterions such as halide, hydroxide, carboxylate, sulfate, phosphate,nitrate, lower alkyl sulfonate, and aryl sulfonate.

The terms “composition” and “formulation” are used interchangeably.

A “subject” to which administration is contemplated refers to a human(i.e., male or female of any age group, e.g., pediatric subject (e.g.,infant, child, or adolescent) or adult subject (e.g., young adult,middle-aged adult, or senior adult)) or non-human animal. In certainembodiments, the non-human animal is a mammal (e.g., primate (e.g.,cynomologus monkey or rhesus monkey), commercially relevant mammal(e.g., cattle, pig, horse, sheep, goat, cat, or dog), or bird (e.g.,commercially relevant bird, such as chicken, duck, goose, or turkey)).In certain embodiments, the non-human animal is a fish, reptile, oramphibian. The non-human animal may be a male or female at any stage ofdevelopment. The non-human animal may be a transgenic animal orgenetically engineered animal. In certain embodiments, the subject is apatient. The term “patient” refers to a human subject in need oftreatment of a disease. In certain embodiments, the term “patient” is ahuman adult over 18 years old in need of treatment of a disease. Incertain embodiments, the term “patient” is a human child no more than 18years old in need of treatment of a disease. In certain embodiments, thepatient is not under regular transfusion (e.g. having had no more than 4transfusion episodes in the 12-month period). In certain embodiments,the patient is under regular transfusion (e.g. having had at least 4transfusion episodes in the 12-month period). In certain embodiments,the subject has undergone splenectomy. In certain embodiments, thesubject has undergone splenectomy and is under regular transfusion. Incertain embodiments, the subject has undergone splenectomy and is notunder regular transfusion.

The term “administer,” “administering,” or “administration” refers toimplanting, absorbing, ingesting, injecting, inhaling, or otherwiseintroducing a compound described herein, or a composition thereof, in oron a subject.

The terms “treatment,” “treat,” and “treating” refer to reversing,alleviating, delaying the onset of, or inhibiting the progress of adisease described herein. In some embodiments, treatment may beadministered after one or more signs or symptoms of the disease havedeveloped or have been observed (i.e., therapeutic treatment). In otherembodiments, treatment may be administered in the absence of signs orsymptoms of the disease. For example, treatment may be administered to asusceptible subject prior to the onset of symptoms (i.e., prophylactictreatment) (e.g., in light of a history of symptoms and/or in light ofexposure to a pathogen). Treatment may also be continued after symptomshave resolved, for example, to delay or prevent recurrence. In certainembodiments, treatment includes delaying onset of at least one symptomof the disorder for a period of time.

The terms “condition,” “disease,” and “disorder” are usedinterchangeably.

An “effective amount” of a compound described herein refers to an amountsufficient to elicit the desired biological response. An effectiveamount of a compound described herein may vary depending on such factorsas the desired biological endpoint, the pharmacokinetics of thecompound, the condition being treated, the mode of administration, andthe age and health of the subject. In certain embodiments, an effectiveamount is a therapeutically effective amount. In certain embodiments,the effective amount is to generate a subject's hemoglobin response of21.5 g/dL increase in Hb concentration from baseline. The subject'sbaseline Hb concentration is the average of all available Hbconcentrations before the treatment with the compound. In certainembodiments, the effective amount is to generate a subject's hemoglobinresponse of ≥1.0 g/dL increase in Hb concentration from baseline. Incertain embodiments, the effective amount is to generate a subject'shemoglobin response of ≥2.0 g/dL increase in Hb concentration frombaseline. In certain embodiments, an effective amount is the amount of acompound described herein in a single dose. In certain embodiments, aneffective amount is the combined amounts of a compound described hereinin multiple doses. In certain embodiments, the effective amount istherapeutically effective amount.

A “therapeutically effective amount” of a compound described herein isan amount sufficient to provide a therapeutic benefit in the treatmentof a condition or to delay or minimize one or more symptoms associatedwith the condition. A therapeutically effective amount of a compoundmeans an amount of therapeutic agent, alone or in combination with othertherapies, which provides a therapeutic benefit in the treatment of thecondition. The term “therapeutically effective amount” can encompass anamount that improves overall therapy, reduces or avoids symptoms, signs,or causes of the condition, and/or enhances the therapeutic efficacy ofanother therapeutic agent. In certain embodiments, a therapeuticallyeffective amount is an amount sufficient for eliciting measurableactivation of wild-type or mutant PKR. In certain embodiments, atherapeutically effective amount is an amount sufficient for regulating2,3-diphosphoglycerate and/or ATP levels in blood in need thereof or fortreating pyruvate kinase deficiency (PKD), hemolytic anemia (e.g.,chronic hemolytic anemia, hereditary non-spherocytic anemia), sicklecell disease, thalassemia (e.g., beta-thalassemia), hereditaryspherocytosis, hereditary elliptocytosis, abetalipoproteinemia (orBassen-Kornzweig syndrome), paroxysmal nocturnal hemoglobinuria,acquired hemolytic anemia (e.g., congenital anemias (e.g.,enzymopathies)), anemia of chronic diseases or treating diseases orconditions that are associated with increased 2,3-diphosphoglyceratelevels (e.g. liver diseases). In certain embodiments, a therapeuticallyeffective amount is an amount sufficient for eliciting measurableactivation of wild-type or mutant PKR and for regulating2,3-diphosphoglycerate levels in blood in need thereof or for treatingpyruvate kinase deficiency (PKD), hemolytic anemia (e.g., chronichemolytic anemia, hereditary non-spherocytic anemia), sickle celldisease, thalassemia (e.g., beta-thalassemia), hereditary spherocytosis,hereditary elliptocytosis, abetalipoproteinemia (or Bassen-Kornzweigsyndrome), paroxysmal nocturnal hemoglobinuria, acquired hemolyticanemia (e.g., congenital anemias (e.g., enzymopathies)), anemia ofchronic diseases or treating diseases or conditions that are associatedwith increased 2,3-diphosphoglycerate levels (e.g., liver diseases). Inone aspect, the therapeutically effective amount is the amount requiredto generate a subject's hemoglobin response of ≥1.0 g/dL (such as ≥21.5g/dL or ≥2.0 g/dL) increase in Hb concentration from baseline. Thesubject's baseline Hb concentration is the average of all available Hbconcentrations within at least two weeks (e.g. 3 weeks, 4 weeks, 5weeks, or 6 weeks) before treatment with a compound described herein. Incertain aspects, the therapeutically effective amount is the amountrequired to reduce the patient's transfusion burden. In one aspect, thetherapeutically effective amount is between 0.01-100 mg/kg bodyweight/day of the provided compound, such as e.g., 0.1-100 mg/kg bodyweight/day. In certain embodiments, the therapeutically effective amountis to reduce the patient's transfusion burden.

As used herein, reduction in transfusion burden means at least 20%reduction in the number of RBC units transfused within at least 5 weeksof treatment. In certain embodiments, the reduction in transfusionburden is ≥33% reduction in the number of RBC units transfused within atleast 5 weeks of treatment. In certain embodiments, reduction oftransfusion burden is observed in at least 10 weeks (e.g., at least 20weeks or at least 24 weeks) of treatment.

As used herein, sickle cell disease (SCD), Hemoglobin SS disease, andsickle cell anemia are used interchangeably. Sickle cell disease (SCD)describes a group of inherited red blood cell disorders. In certainembodiments, subjects with SCD have abnormal hemoglobin, calledhemoglobin S or sickle hemoglobin, in their red blood cells. In certainembodiments, people having SCD have at least one abnormal genes causingthe body to make hemoglobin S. In certain embodiments, people having SCDhave two hemoglobin S genes, Hemoglobin SS.

Thalassemia is an inherited blood disorder in which the body makes anabnormal form of hemoglobin. In certain embodiments, the abnormal formof hemoglobin results in deficiency of either alpha or beta globin. Incertain embodiments, the disorder results in large numbers of red bloodcells being destroyed, which leads to anemia. In certain embodiments,the thalassemia is alpha thalassemia. In certain embodiments, thethalassemia is beta thalassemia.

The term “activator” as used herein means an agent that (measurably)increases the activity of wild type pyruvate kinase R (wt PKR) or causeswild type pyruvate kinase R (wt PKR) activity to increase to a levelthat is greater than wt PKR's basal levels of activity or an agent that(measurably) increases the activity of a mutant pyruvate kinase R (mPKR)or causes mutant pyruvate kinase R (mPKR) activity to increase to alevel that is greater than that mutant PKR's basal levels of activity,for examples, to a level that is 20%, 40%, 50%, 60%, 70%, 80%, 90% or100% of the activity of wild type PKR.

The term “packed red blood cells” or PRBCs as used herein refer to redblood cells made from a unit of whole blood by centrifugation andremoval of most of the plasma. In certain embodiments, a PRBC unit has ahematocrit of at least about 95%. In certain embodiments, a PRBC unithas a hematocrit of at least about 90%, 80%, 70%, 60%, 50%, 40%, 30%,20%, or 10%.

The term “ex vivo” referring to a method as used herein means that themethod takes place outside an organism. For example, a cell (e.g., redblood cells), a tissue or blood (containing at least red blood cells,plasma and hemoglobin) may be extracted from the organism to becontacted with one or more compounds provided herein or apharmaceutically acceptable salt thereof or a pharmaceutical compositionthereof, optionally under artificially controlled conditions (e.g.,temperature).

The term “in vitro” referring to a method as used herein means that themethod takes place outside an organism and is contained within anartificial environment. For example, a cell (e.g., red blood cells), atissue or blood (containing at least red blood cells, plasma andhemoglobin) may be extracted from the organism to be contacted with oneor more compounds provided herein or a pharmaceutically acceptable saltthereof or a pharmaceutical composition thereof, in a contained,artificial environment (e.g., a culture system), such as in a test tube,in a culture, in flask, in a microtiter plate, on a Petri dish, and thelike.

Compounds

Described herein are compounds and compositions that activate wild typePKR and/or mutant PKRs such as those described herein. In oneembodiment, provided is a compound of Formulas (I)-(V), or apharmaceutically acceptable salt thereof, or a pharmaceuticalcomposition comprising a compound of Formulas (I)-(V), or apharmaceutically acceptable salt thereof.

In a first embodiment of the invention, provided is a compoundrepresented by Formula (I):

or a pharmaceutically acceptable salt thereof, wherein:

R¹ is hydrogen, an optionally substituted alkyl, an optionallysubstituted haloalkyl, an optionally substituted alkenyl, an optionallysubstituted alkynyl, an optionally substituted cycloalkyl, an optionallysubstituted heterocyclyl, an optionally substituted aryl, —OR^(o1),—C(═O)R^(c1), or a nitrogen protecting group; wherein:

-   -   R^(o1) is hydrogen, optionally substituted alkyl, or an oxygen        protecting group;    -   R^(c1) is optionally substituted alkyl or —N(R^(cn))₂, wherein        each instance of

R^(cn) is independently hydrogen, —C₁₋₆ alkyl, or a nitrogen protectinggroup;

R² and Q are each independently an optionally substituted 5- or6-membered monocyclic heteroaryl;

R^(a) and R^(b) are each independently hydrogen, a halogen, —CN, —NO₂,—N₃, an optionally substituted alkyl, —OR^(o3), —N(R^(n1))₂,—C(═O)N(R^(n1))₂, or C(═O)R^(c2); or alternatively R^(a) and R^(b) canbe taken together with the carbon atom to which they are attached toform an optionally substituted cycloalkyl or an optionally substitutedheterocyclyl; wherein:

-   -   each instance of R^(n1) is independently hydrogen, an optionally        substituted —C₁-C₆ alkyl, or a nitrogen protecting group;    -   R^(o3) is hydrogen, an optionally substituted —C₁-C₆ alkyl, or        an oxygen protecting group; and    -   R^(c2) is an optionally substituted —C₁-C₆ alkyl; and

R^(j) and R^(k) are each independently hydrogen, a halogen, —CN,—OR^(o7), —N(R^(n5))₂, —N(R^(n5))C(═O) R^(c5), —C(═O)N(R^(n5))₂,—C(═O)R^(c5), —C(═O)OR^(o7), —SR^(js), —S(═O)₂R^(js), —S(═O)R^(js), oran optionally substituted —C₁-C₆ alkyl; or alternatively R^(j) and R^(k)can be taken together with the carbon atom to which they are attached toform C═O, an optionally substituted C₁-C₆ monocyclic cycloalkyl ring, oran optionally substituted C₃-C₆ monocyclic heterocyclyl ring; wherein:

-   -   each instance of R^(n5) is independently hydrogen, an optionally        substituted —C₁-C₆ alkyl, —OR^(o8), or a nitrogen protecting        group, wherein R^(o8) is hydrogen, an optionally substituted        —C₁-C₆ alkyl, or an oxygen protecting group;    -   each instance of R^(o7) is independently hydrogen, an optionally        substituted —C₁-C₆ alkyl, or an oxygen protecting group;    -   each instance of R^(c5) is independently an optionally        substituted —C₁-C₆ alkyl; and    -   each instance of R^(js) is independently an optionally        substituted —C₁-C₆ alkyl, an optionally substituted C₆₋₁₂ aryl,        an optionally substituted heteroaryl, or a sulfur protecting        group.

In a second embodiment of the invention, provided is a compoundrepresented by Formula (I) or a pharmaceutically acceptable saltthereof, wherein the 5- or 6-membered monocyclic heteroaryl representedby R² is optionally substituted at each substitutable ring carbon atomby R^(p) and optionally substituted at each substitutable ring nitrogenatom by R^(n6); wherein:

each instance of R^(p) is independently hydrogen, a halogen, —CN, —NO₂,—N₃, an optionally substituted alkyl, an optionally substituted alkenyl,an optionally substituted alkynyl, an optionally substituted cycloalkyl,an optionally substituted aryl, an optionally substituted heterocyclyl,an optionally substituted heteroaryl, —OR^(o6), —SR^(s2), —N(R^(n3))₂,—C(═O)N(R^(n3))₂, —N(R^(n3))C(═O)R^(c4), —C(═O)R^(c4), —C(═O)OR^(o6),—OC(═O)R^(c4), —S(═O)R^(s2), —S(═O)₂R^(s2), —S(═O)OR^(o6),—OS(═O)R^(c4), —S(═O)₂R^(o6), —OS(═O)₂R^(c4), —S(═O)N(R^(n3))₂,—S(═O)₂N(R^(n3))₂, —N(R^(n3))S(═O)R^(s2), —N(R^(n3))S(═O)₂R^(s2),—N(R^(n3))C(═O)OR^(o6), —OC(═O)N(R^(n3))₂, —N(R^(n3))C(═O)N(R^(n3))₂,—N(R^(n3))S(═O)N(R^(n3))₂, —N(R^(n3))S(═O)₂N(R^(n3))₂,—N(R^(n3))S(═O)OR^(o6), —N(R′)S(═O)₂OR^(o6), —OS(═O)N(R^(n3))₂, or—OS(═O)₂N(R^(n3))₂; or alternatively two instances of R^(p) attached tothe same or adjacent carbon atoms, can be taken together with the carbonatom(s) to which they are attached to form an optionally substitutedcycloalkyl or a heterocycloalkyl; wherein:

-   -   each instance of R^(n3) is independently hydrogen, an optionally        substituted —C₁-C₆ alkyl, or a nitrogen protecting group;    -   each instance of R^(o6) is independently hydrogen, an optionally        substituted —C₁-C₆ alkyl, or an oxygen protecting group; and    -   each instance of R^(c4) is an optionally substituted —C₁-C₆        alkyl;    -   each instance of R^(c2) is independently an optionally        substituted —C₁-C₆ alkyl or a sulfur protecting group; and

R^(n6) is hydrogen, an optionally substituted —C₁-C₆ alkyl, or anitrogen protecting group;

wherein the remaining variables are as defined in the first embodiment.

In a third embodiment of the invention, provided is a compoundrepresented by Formula (I) or a pharmaceutically acceptable saltthereof, wherein the 5- or 6-membered monocyclic heteroaryl representedby R² is selected from one of the following:

wherein

each instance of R^(nc) and R^(nd) is independently hydrogen, optionallysubstituted —C₁-C₆ alkyl, or a nitrogen protecting group;

p is 0, 1, 2, 3, or 4, as valency permits;

wherein the remaining variables are as defined in the first or secondembodiments.

In a fourth embodiment of the invention, provided is a compoundrepresented by Formula (I) or a pharmaceutically acceptable saltthereof, wherein the 5- or 6-membered monocyclic heteroaryl representedby R² is selected from one of the following:

wherein the remaining variables are as defined in the first, second, orthird embodiments.

In a fifth embodiment of the invention, provided is a compoundrepresented by Formula (I) or a pharmaceutically acceptable saltthereof, wherein the 5- or 6-membered monocyclic heteroaryl representedby R² is selected from one of the following:

Wherein the remaining variables are as defined in the first, second,third, or fourth embodiments.

In a sixth embodiment of the invention, provided is a compoundrepresented by Formula (II):

or a pharmaceutically acceptable salt thereof, wherein q is 0, 1, 2, or3; wherein the remaining variables are as defined in the first, second,third, fourth, or fifth embodiments. In certain embodiments, provided isa compound represented by one of the following formulae:

a pharmaceutically acceptable salt thereof.

In a seventh embodiment of the invention, provided is a compoundrepresented by Formula (III):

or a pharmaceutically acceptable salt thereof, wherein q is 0, 1, 2, or3; wherein the remaining variables are as defined in the first, second,third, fourth, fifth, or sixth embodiments:

In an eighth embodiment of the invention, provided is a compoundrepresented by Formula (IV):

or a pharmaceutically acceptable salt thereof, wherein q is 0, 1, 2, or3; wherein the remaining variables are as defined in the first, second,third, fourth, fifth, sixth, or seventh embodiments.

In a ninth embodiment of the invention, provided is a compoundrepresented by Formula (V):

or a pharmaceutically acceptable salt thereof, wherein q is 0, 1, 2, or3; wherein the remaining variables are as defined in the first, second,third, fourth, fifth, sixth, seventh, or eighth embodiments.

In a tenth embodiment of the invention, provided is a compoundrepresented by Formula (II), (III), (IV), or (V) or a pharmaceuticallyacceptable salt thereof, wherein R^(n6) is hydrogen or a —C₁₋₄ alkyl;wherein the remaining variables are as defined in the second, third,fourth, fifth, sixth, seventh, eighth, or ninth embodiments.

In an eleventh embodiment of the invention, provided is a compoundrepresented by Formula (II), (III), (IV), or (V) or a pharmaceuticallyacceptable salt thereof, wherein each instance of R^(p) is independentlyhydrogen, halogen, optionally substituted C₁₋₄ alkyl, —CN, —NO₂, —N₃,—OR^(o4), —N(R^(n2))₂, —C(═O)N(R^(n2))₂, —C(═O)R^(c3), or —C(═O)OR^(o4);wherein the remaining variables are as defined in the second, third,fourth, fifth, sixth, seventh, eighth, ninth, or tenth embodiments.

In a twelfth embodiment of the invention, provided is a compoundrepresented by Formula (I), (II), (III), (IV), or (V) or apharmaceutically acceptable salt thereof, wherein the 5- or 6-memberedmonocyclic heteroaryl represented by Q is selected from the following:

wherein:

each instance of R^(n) is independently hydrogen, a halogen, —CN, —NO₂,—N₃, an optionally substituted alkyl, an optionally substituted alkenyl,an optionally substituted alkynyl, an optionally substituted cycloalkyl,an optionally substituted aryl, an optionally substituted heterocyclyl,an optionally substituted heteroaryl, —OR⁴, —SR^(s1), —N(R^(n2))₂,—C(═O)N(R^(n2))₂, —N(R^(n2))C(═O)R^(c3), —C(═O)R^(c3), —C(═O)OR^(o4),—OC(═O)R^(c3), —S(═O)R^(s1), —S(═O)₂R^(s1), —S(═O)OR^(o4),—OS(═O)R^(c3), —S(═O)₂OR^(o4), —OS(═O)₂R^(c3), —S(═O)N(R^(n2))₂,—S(═O)₂N(R^(n2))₂, —N(R^(n2))S(═O)R^(s1), —N(R^(n2))S(═O)₂R^(s1),—N(R^(n2))C(═O)OR^(o4), —OC(═O)N(R^(n2))₂, —N(R^(n2))C(═O)N(R^(n2))₂,—N(R^(n2))S(═O)N(R^(n2))₂, —N(R^(n2))S(═O)₂N(R^(n2))₂,—N(R^(n2))S(═O)OR^(o4), —N(R^(n2))S(═O)₂OR^(o4), —OS(═O)N(R^(n2))₂, or—OS(═O)₂N(R^(n2))₂; or two instances of R^(n) attached to the same oradjacent carbon atoms, taken together with the carbon atoms to whichthey are attached to form an optionally substituted cycloalkyl or aheterocycloalkyl; wherein:

-   -   each instance of R^(n2) is independently hydrogen, an optionally        substituted —C₁-C₆ alkyl, or a nitrogen protecting group;    -   each instance of R⁴ is independently hydrogen, an optionally        substituted —C₁-C₆ alkyl, or an oxygen protecting group;    -   each instance of R^(c3) is independently an optionally        substituted —C₁-C₆ alkyl;    -   each instance of R^(s1) is independently an optionally        substituted —C₁-C₆ alkyl or a sulfur protecting group;    -   n is 0, 1, 2, or 3, as valency permits; and    -   each of R^(na), R^(nb), and R^(nd) is independently hydrogen, an        optionally substituted —C₁-C₆ alkyl, or a nitrogen protecting        group.

wherein the remaining variables are as defined in the first, second,third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, or eleventhembodiments.

In a thirteenth embodiment of the invention, provided is a compoundrepresented by Formula (I), (II), (III), (IV), or (V) or apharmaceutically acceptable salt thereof, wherein the 5- or 6-memberedmonocyclic heteroaryl represented by Q is selected from the following:

wherein the remaining variables are as defined in the first, second,third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, ortwelfth embodiments.

In a fourteenth embodiment of the invention, provided is a compoundrepresented by Formula (I), (II), (III), (IV), or (V) or apharmaceutically acceptable salt thereof, wherein the 5- or 6-memberedmonocyclic heteroaryl represented by Q is selected from the following:

wherein the remaining variables are as defined in the first, second,third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh,twelfth, or thirteenth embodiments.

In a fifteenth embodiment of the invention, provided is a compoundrepresented by Formula (I), (II), (III), (IV), or (V) or apharmaceutically acceptable salt thereof, wherein Q is

or wherein the remaining variables are as defined in the first, second,third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh,twelfth, thirteenth, or fourteenth embodiments. In further embodiments,provided is a compound represented by Formula (I), (II), (III), (IV), or(V) or a pharmaceutically acceptable salt thereof, wherein Q is

wherein the remaining variables are as defined in the first, second,third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh,twelfth, thirteenth, or fourteenth embodiments.

In a sixteenth embodiment of the invention, provided is a compoundrepresented by Formula (I), (II), (III), (IV), or (V) or apharmaceutically acceptable salt thereof, wherein Q is

wherein the remaining variables are as defined in the first, second,third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh,twelfth, thirteenth, or fourteenth embodiments. In further embodiments,provided is a compound represented by Formula (I), (II), (Ill), (IV), or(V) or a pharmaceutically acceptable salt thereof, wherein Q is

wherein the remaining variables are as defined in the first, second,third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh,twelfth, thirteenth, or fourteenth embodiments. In certain embodiments,R² and Q are the same. In certain embodiments, R² and Q are different.

In a seventeenth embodiment of the invention, provided is a compoundrepresented by Formula (I), (II), (III), (IV), or (V) or apharmaceutically acceptable salt thereof, wherein R^(na) is hydrogen or—C₁₋₄ alkyl; wherein the remaining variables are as twelfth, thirteenth,fourteenth, or fifteenth embodiments.

In a seventeenth embodiment of the invention, provided is a compoundrepresented by Formula (I), (II), (III), (IV), or (V) or apharmaceutically acceptable salt thereof, wherein R^(nb) is hydrogen or—C₁₋₄ alkyl; wherein the remaining variables are as twelfth, thirteenth,fourteenth, or fifteenth embodiments.

In a seventeenth embodiment of the invention, provided is a compoundrepresented by Formula (I), (II), (III), (IV), or (V) or apharmaceutically acceptable salt thereof, wherein R^(nd) is hydrogen or—C₁₋₄ alkyl; wherein the remaining variables are as twelfth, thirteenth,fourteenth, or fifteenth embodiments.

In an eighteenth embodiment of the invention, provided is a compoundrepresented by Formula (I), (II), (III), (IV), or (V) or apharmaceutically acceptable salt thereof, wherein each instance of R^(n)is independently hydrogen, halogen, optionally substituted C₁₋₄ alkyl,—CN, —NO₂, —N₃, —OR^(o4), —N(R²)₂, —C(═O)N(R^(n2))₂, —C(═O)R^(c3), or—C(═O)OR⁴; wherein the remaining variables are as defined in thetwelfth, thirteenth, fourteenth, fifteenth, sixteenth, or seventeenthembodiments.

In a nineteenth embodiment of the invention, provided is a compoundrepresented by Formula (I), (II), (III), (IV), or (V) or apharmaceutically acceptable salt thereof, wherein R¹ is hydrogen or a—C₁-C₄ alkyl; wherein the remaining variables are as defined in thefirst, second, third, fourth, fifth, sixth, seventh, eighth, ninth,tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, sixteenth,seventeenth, or eighteenth embodiments.

In a twentieth embodiment of the invention, provided is a compoundrepresented by Formula (I), (II), (III), (IV), or (V) or apharmaceutically acceptable salt thereof, wherein R¹ is methyl; whereinthe remaining variables are as defined in the first, second, third,fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth,thirteenth, fourteenth, fifteenth, sixteenth, seventeenth, or eighteenthembodiments.

In a twenty-first embodiment of the invention, provided is a compoundrepresented by Formula (I), (II), (I), (IV), or (V) or apharmaceutically acceptable salt thereof, wherein R^(j) and R^(k) areeach independently hydrogen, a halogen, —OR^(o7), or a —C₁-C₄ alkyl; oralternatively R^(j) and R^(k) are joined together to form ═O wherein theremaining variables are as defined in the first, second, third, fourth,fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth,thirteenth, fourteenth, fifteenth, sixteenth, seventeenth, eighteenth,nineteenth, or twentieth embodiments.

In a twenty-second embodiment of the invention, provided is a compoundrepresented by Formula (I), (II), (III), (IV), or (V) or apharmaceutically acceptable salt thereof, wherein R^(j) and R^(k) areeach hydrogen; wherein the remaining variables are as defined in thefirst, second, third, fourth, fifth, sixth, seventh, eighth, ninth,tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, sixteenth,seventeenth, eighteenth, nineteenth, twentieth, or twenty-firstembodiments.

In a twenty-third embodiment of the invention, provided is a compoundrepresented by Formula (I), (II), (II), (IV), or (V) or apharmaceutically acceptable salt thereof, wherein R^(a) and R^(b) areeach hydrogen; wherein the remaining variables are as defined in thefirst, second, third, fourth, fifth, sixth, seventh, eighth, ninth,tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, sixteenth,seventeenth, eighteenth, nineteenth, twentieth, twenty-first, ortwenty-second embodiments.

In a twenty-fourth embodiment of the invention, provided is a compoundrepresented by Formula (I), (II), (III), (IV), or (V) or apharmaceutically acceptable salt thereof, wherein q is 0 or 1; whereinthe remaining variables are as defined in the fifth, sixth, seventh,eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth,fifteenth, sixteenth, seventeenth, eighteenth, nineteenth, twentieth,twenty-first, twenty-second, or twenty-third embodiments.

In a twenty-fifth embodiment of the invention, provided is a compoundrepresented by Formula (I), (I), (III), (IV), or (V) or apharmaceutically acceptable salt thereof, wherein n is 0 or 1; whereinthe remaining variables are as defined in the twelfth, thirteenth,fourteenth, fifteenth, sixteenth, seventeenth, eighteenth, nineteenth,twentieth, twenty-first, twenty-second, twenty-third, or twenty-fourthembodiments.

In one embodiment, the compound of pharmaceutically acceptable saltthereof is selected from the compounds of Formulas (I)-(V), in theExamples, and in Table 1 and FIG. 1.

Compounds described herein are useful as activators of PKR mutantshaving lower activities compared to the wild type, thus are useful formethods of the present invention. Such mutations in PKR can affectenzyme activity (catalytic efficiency), regulatory properties(modulation by fructose bisphosphate (FBP)/ATP), and/or thermostabilityof the enzyme. Examples of such mutations are described in Valentini etal, JBC 2002. Some examples of the mutants that are activated by thecompounds described herein include G332S, G364D, T384M, R479H, R479K,R486W, R532W, K410E, R510Q, and R490W. Without being bound by theory,compounds described herein affect the activities of PKR mutants byactivating FBP non-responsive PKR mutants, restoring thermostability tomutants with decreased stability, or restoring catalytic efficiency toimpaired mutants. The activating activity of the present compoundsagainst PKR mutants may be tested following a method described inExamples 11-17. Compounds described herein are also useful as activatorsof wild type PKR.

In an embodiment, to increase the lifetime of the red blood cells, acompound, composition or pharmaceutical composition described herein isadded directly to whole blood or packed red blood cells extracorporeallyor be provided to the patient directly (e.g., by i.p., i.v., i.m., oral,inhalation (aerosolized delivery), transdermal, sublingual and otherdelivery routes). Without being bound by theory, compounds describedherein increase the lifetime of the RBCs, thus counteract aging ofstored blood, by impacting the level of 2,3-DPG and/or ATP from theblood. A decrease in the level of 2, 3-DPG concentration induces aleftward shift of the oxygen-hemoglobin dissociation curve and shiftsthe allosteric equilibrium to the R, or oxygenated state, thus producinga therapeutic inhibition of the intracellular polymerization thatunderlies sickling by increasing oxygen affinity due to the 2,3-DPGdepletion, thereby stabilizing the more soluble oxy-hemoglobin.Accordingly, in one embodiment, compounds and pharmaceuticalcompositions described herein are useful as antisickling agents. Inanother embodiment, to regulate 2,3-diphosphoglycerate, a compound,composition or pharmaceutical composition described herein is addeddirectly to whole blood or packed red blood cells extracorporeally or beprovided to the patient directly (e.g., by i.p., i.v., i.m., oral,inhalation (aerosolized delivery), transdermal, sublingual and otherdelivery routes). In another embodiment, a compound, composition orpharmaceutical composition described herein can increase the level ofATP and help to protect the cells from reactive oxygen species (MolCell. 2012 Oct. 26; 48(2): 158-167).

In Table 1, a compound described herein may have an AC₅₀ of wild typePKR, PKR K410E or PKR 510Q. “A” refers to an AC₅₀ less than 0.300 μM;“B” refers to an AC₅₀ from 0.301 μM to 0.800 μM; and “C” refers to anAC₅₀ greater than 0.800 μM. The AC₅₀ of wild type PKR for certaincompounds was additionally determined in a cell-based ATP assay. “AA”refers to an AC₅₀ less than or equal to 1 μM and “BB” refers to an AC₅₀more than 1 μM.

TABLE 1 Activation of wild type and mutant PKR by exemplary compoundsPKR PKR PKR Cell based WT K410E 510Q ATP assay Compound AC₅₀ AC₅₀ AC₅₀AC₅₀

A A A AA

A A A AA

A A A AA

A A A AA

A A A AA

A A A AA

A A A AA

A A A AA

A A A AA

A A A AA

A A A AA

A A A AA

A A A AA

A A A AA

A A A AA

A A A AA

A A A AA

A A A AA

A A A AA

A A A AA

A A A AA

A A A AA

A A A AA

A A A AA

A A A AA

A A A AA

A A A AA

A A A AA

A A A AA

A A A AA

C C C BB

A A A AA

A A A AA

A A A AA

A A A AA

A A A AA

B A A AA

A A A AA

A A A AA

A A A AA

A A A AA

A A A AA

A A A AA

A A A AA

A A A AA

A A A AA

A A A AA

A A A AA

A A A AA

A A A AA

A A A AA

A A A AA

A A A AA

A A A AA

A A A AA

A A A AA

A A A AA

A A A AA

A A A AA

A A A AA

A A A AA

A A A AA

A A B AA

C C C BB

A A A AA

A A A AA

A A A AA

A A A AA

A A A AA

A A A AA

A A A AA

A A A AA

A A A AA

A A A AA

A A A AA

A A A AA

C C C BB

A A A AA

A A A AA

A A A AA

A A A AA

A A A AA

A A A AA

A A A AA

A A A AA

A A A AA

A A A AA

A A A AA

A A A AA

A A A AA

B B C BB

A A A AA

A A A AA

A A A AA

A A A AA

A A A AA

A A A AA

C C C BB

A A A AA

A A A AA

A A A AA

A A A AA

A A B AA

A A A AA

A A A AA

A A A AA

A A A AA

A A A AA

C C A BB

A A A AA

A A A AA

A A A AA

A A A AA

A A A AA

A A A AA

A A A AA

A A A AA

A A A AA

A A B AA

A A B BB

A A A AA

A A A AA

A A A AA

A A A AA

A A A AA

A A A AA

A A A AA

C B C BB

A A A AA

A A A AA

A A A AA

A A A AA

A A A AA

A A A AA

A A A AA

A A A AA

A A A AA

A A A AA

A A A AA

B A A AA

B A C AA

A A A AA

A A A AA

A A A AA

A A A AA

A A A AA

A A A AA

A A A AA

A A A AA

A A A AA

A A A AA

A A B AA

A A A AA

B B B BB

A A A AA

A A A AA

A A A AA

A A A AA

A A A AA

C C C BB

A A A AA

B B B BB

A A A AA

C C A BB

C C C BB

B B C BB

A A A AA

A A A AA

C C C BB

C* C* C* AA

B B A BB

B B B BB

A A A AA

A B A AA

A A A AA

A A A AA

C C C BB

A A A AA

A A A AA

A A A AA

A A A AA

A A A AA

C C C BB

C* C* C* AA

B* A* A* AA

C C C BB

B B A AA

C C C No Fit

C C C No Fit

No Fit No Fit No Fit No Fit

A A A AA

A A A AA

B C B AA *indicates the value is based on the average of multiple testedresults.

The compounds described herein can be made using a variety of synthetictechniques as set forth in the Examples. Synthetic chemistrytransformations and protecting group methodologies (protection anddeprotection) useful in synthesizing the compounds described herein areknown in the art and include, for example, those such as described in R.Larock, Comprehensive Organic Transformations, VCH Publishers (1989); T.W. Greene and P. G. M. Wuts, Protective Groups in Organic Synthesis, 2d.Ed., John Wiley and Sons (1991); L. Fieser and M. Fieser, Fieser andFieser's Reagents for Organic Synthesis, John Wiley and Sons (1994); andL. Paquette, ed., Encyclopedia of Reagents for Organic Synthesis, JohnWiley and Sons (1995), and subsequent editions thereof.

Certain activator compounds useful as PKR wild type and/or mutantactivators are those that demonstrate specificity and activation of PKRenzyme (wild type and/or a mutant enzyme) in the absence of FBP to alevel greater than that of 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60,65, 70, 75, 80, 85, 90, 95, 99, or 100% in the presence of FBP.

Synthesis of the Compounds of the Invention

In some embodiments, compounds of Formula (I) can be prepared usingmethods illustrated in Scheme 1. Thiazolyl aldehyde of formula S1 reactswith ethyl azidoacetate under nucleophilic addition conditions (e.g. abase) in an appropriate solvent (e.g. ethanol) to give intermediates offormula S2. The hydroxyl group of formula S2 can be converted to aleaving group and subject to elimination to give formula S3. Cyclizationand subsequent functionalization of the amino group provides bicycliccompound of formula S5, which undergoes nucleophilic displacement withsodium methanethiolate, followed by oxidation to give formula S7.Further cyclization of formula S7 in the presence of hydrazine, followedby nucleophilic displacement with LG¹-CH₂-Q¹ in the presence of a baseprovides intermediates of formula S9. The sulfur group in formula S9 canbe oxidized to sulfinyl or sulfonyl to provide formula S10 or S11, whichis a substrate for further nucleophilic displacement to generate ageneral formula S12. As used herein, X¹ is a leaving group as definedherein. In certain embodiments, X¹ is halogen, alkanesulfonyloxy,arenesulfonyloxy, diazonium, alkyl diazenes, aryl diazenes, alkyltriazenes, aryl triazenes, nitro, alkyl nitrate, aryl nitrate, alkylphosphate, aryl phosphate, alkyl carbonyl oxy, aryl carbonyl oxy,alkoxcarbonyl oxy, aryoxcarbonyl oxy ammonia, alkyl amines, aryl amines,hydroxyl group, alkyloxy group, aryloxy group; LG¹ is a leaving group asdefined herein; Q¹ is optionally substituted cycloalkyl, optionallysubstituted heterocyclyl, optionally substituted aryl, or optionallysubstituted heteroaryl (e.g. optionally substituted 5- or 6-memberedmonocyclic heteroaryl); and Nu¹ is a nucleophile as defined herein (e.g.optionally substituted 5- or 6-membered monocyclic heteroaryl alkylene).Nu¹ of compound of formula S12 can be further converted to otherfunctionalities with standard chemical transformations. R¹ is as definedin the first embodiment. In certain embodiments, Q¹ is optionallysubstituted heteroaryl.

In some embodiments, compounds of Formula (I) can be prepared usingmethods shown in Scheme 2. Similar to Scheme 1, formula S21 can beprepared from thiazole aldehyde of formula S13. Halogenation of formulaS21 gives formula S22, which can undergo an organo coupling reactionwith an alkyl metal, alkenyl metal, alkynyl metal, aryl metal,heteroaryl metal, heterocyclyl metal, or cycloalkyl metal to give acompound of formula 223. As used herein, X³ is a halogen; R¹ is asdefined in the first embodiment of the invention; LG² is a leaving groupas defined herein; Q² is optionally substituted cycloalkyl, optionallysubstituted heterocyclyl, optionally substituted aryl, or optionallysubstituted heteroaryl (e.g. optionally substituted 5- or 6-memberedmonocyclic heteroaryl); M¹ is a metal (e.g. Li, Na, K, Mg, Zn, Sn, B,Pd, Si, Cu etc.), X⁴ is halogen or alkyl sulfonic acid ester or an arylsulfonic acid ester; R^(r1) is optionally substituted alkyl, optionallysubstituted alkenyl, optionally substituted alkynyl, optionallysubstituted cycloalkyl, optionally substituted heterocyclyl, optionallysubstituted aryl, or optionally substituted heteroaryl. In certainembodiments, the organo coupling reaction is Negishi reaction; X³ is I;and M¹ is Zn.

Compounds of formula S22 and S23 are useful intermediates to introducemore functionalities at X³ and/or R^(r1) position (Scheme 3). In certainembodiments, the compound of formula 23-i can be further oxidized toform formula S24. Nucleophilic addition of S24 with an appropriatenucleophile generates a compound of 525. In another embodiment,compounds of formula S22 can be coupled with vinyl metal to introducethe vinyl group to the thiazole ring. Oxidation of the vinyl groupfollowed by nucleophilic addition provides a compound of formula 528. Asused herein, Nu² is a nucleophile. In certain embodiments, R^(r1) isoptionally substituted 5- or 6-membered monocyclic heteroarylalkyleneand Q² is optionally substituted 5- or 6-membered monocyclic heteroaryl.

As used herein, R^(r2) is optionally substituted alkyl, optionallysubstituted alkenyl, optionally substituted alkynyl, optionallysubstituted cycloalkyl, optionally substituted heterocyclyl, optionallysubstituted aryl, or optionally substituted heteroaryl; and Q² is asdefined in Scheme 2. In certain embodiments, R^(r2) and Q² are eachindependently optionally substituted 5- or 6-membered monocyclicheteroaryl.

As used herein, a nucleophile is a chemical species that donates anelectron pair to an electrophile to form a chemical bond in relation toa reaction. All molecules or ions with a free pair of electrons or atleast one pi bond can act as nucleophiles. Exemplary nucleophilescomprise at least one group possessing nucleophilic functionality, forexample, an alpha carbon (e.g. the carbon adjacent to carbonyl,sulfonyl, sulfinyl, aryl group, or heteroaryl), a thiol group, ahydroxyl group, a primary amine group, a secondary amine group, ahalide, cyanide, azide, alcoxide, organic metal, or inorganic base.

In some embodiments, compounds as described herein can be prepared usingmethods shown in Scheme 4. Nucleophilic displacement of formulae S30 andS33 with a secondary cyclic amine provides formulae S31 and S34respectively. Organo-coupling reactions (e.g. Suzuki coupling,Stillecoupling etc.) of compound S35 provide a compound of formulaeS36(i)-(ii).

As used herein,

represents a 5- or 6-membered monocyclic heteroaryl ring A with anitrogen as a ring atom. As used herein,

represents a 5- or 6-membered monocyclic heteroaryl ring B with thepoint of attachment on the carbon ring atom.

R¹ is as defined in the first embodiment. X⁴ is halogen or -OTf. M⁴ isan organic metal with appropriate ligands if needed (organic orinorganic) as valency permits. Exemplified M⁴ includes, but is notlimited to organic Li, Sn, B (e.g. boronic acids and boronic esters),Zn, Mg, Si, Pd, and Cu.

Methods of Treatment

In one embodiment, provided is a method for treating a disease,condition or disorder as described herein (e.g., treating) comprisingadministering a compound, a pharmaceutically acceptable salt of acompound or pharmaceutical composition comprising a compound describedherein (e.g., a compound of Formulas (I)-(V), in the Examples, and inTable 1 and FIG. 1.

The compounds and compositions described herein can be administered tocells in culture, e.g. in vitro or ex vivo, or to a subject, e.g., invivo, to treat, and/or diagnose a variety of disorders, including thosedescribed herein below.

In one embodiment of the invention provided is a method for increasingthe lifetime of red blood cells (RBCs) in need thereof comprisingcontacting blood with an effective amount of (1) a compound describedherein (e.g., Formulas (I)-(V), in the Examples, and in Table 1 and FIG.1, or a pharmaceutically acceptable salt thereof; (2) a pharmaceuticallyacceptable composition comprising a compound described herein or apharmaceutically acceptable salt thereof, and a pharmaceuticallyacceptable carrier.

In a further embodiment the compound or pharmaceutical composition isadded directly to whole blood or packed red blood cells (e.g.extracorporeally). In another embodiment, the compound or pharmaceuticalcomposition is administered to a subject in need thereof.

In one embodiment of the invention provided is a method for regulating2,3-diphosphoglycerate levels in blood in need thereof contacting bloodwith an effective amount of (1) a compound described herein (e.g., acompound of Formulas (I)-(V), in the Examples, and in Table 1 and FIG.1, or a pharmaceutically acceptable salt thereof; (2) a pharmaceuticallyacceptable composition comprising a compound described herein or apharmaceutically acceptable salt thereof, and a pharmaceuticallyacceptable carrier.

In one embodiment of the invention provided is a method for treatingsickle cell disease comprising administering to a subject in needthereof with an effective amount of (1) a compound described herein(e.g., a compound of Formulas (I)-(V), in the Examples, and in Table 1and FIG. 1, or a pharmaceutically acceptable salt thereof; (2) apharmaceutically acceptable composition comprising a compound describedherein or a pharmaceutically acceptable salt thereof, and apharmaceutically acceptable carrier.

As used here, sickle cell disease (SCD), Hemoglobin SS disease, andsickle cell anemia are used interchangeably. Sickle cell disease (SCD)describes a group of inherited red blood cell disorders. In certainembodiments, subjects with SCD have abnormal hemoglobin, calledhemoglobin S or sickle hemoglobin, in their red blood cells. In certainembodiments, a subject having SCD has at least one abnormal genescausing the body to make hemoglobin S. In certain embodiments, a subjecthaving SCD has two hemoglobin S genes, Hemoglobin SS.

In one embodiment of the invention provided is a method of treatingpyruvate kinase deficiency (PKD) in a subject comprising administeringto the subject an effective amount of (1) a compound described herein(e.g., a compound of Formulas (I)-(V), in the Examples, and in Table 1and FIG. 1, or a pharmaceutically acceptable salt thereof; (2) apharmaceutically acceptable composition comprising a compound describedherein or a pharmaceutically acceptable salt thereof, and apharmaceutically acceptable carrier.

As described herein, PKD is a deficiency of PKR. In certain embodiments,the deficiency of PKR is associated with a PKR mutation. In certainembodiments, PKD refers to presence of at least 2 mutant alleles in thePKLR gene. In certain embodiments, at least 1 of the at least 2 mutantallels in the PKLR gene is a missense mutation. In certain embodiments,a PKD patient has an Hb concentration less than or equal to 10.0 g/dL.In certain embodiments, the patient is not under regularly transfusion(e.g. having had no more than 4 transfusion episodes in the 12-monthperiod). In certain embodiments, the patient is under regularlytransfusion (e.g. having had at least 4 transfusion episodes in the12-month period). In certain embodiments, the patient is under aregularly transfusion having at least 6 transfusion episodes in the12-month period. In certain embodiments, the patient under regulartransfusion has hemoglobin (Hb)≤12.0 g/dL (if male) or ≤11.0 g/dL (iffemale) example 15 In certain embodiments, the patient has undergonesplenectomy.

In an embodiment, the PKR mutation is selected from the group consistingof A31V, A36G, G37Q, R40W, R40Q, L73P, S80P, P82H, R86P, 190N, T93I,G95R, M107T, G111R, A115P, S120F, H121Q, S130P, S130Y, V134D, R135D,A137T, G143S, I153T, A154T, L155P, G159V, R163C, R163L, T164N, G165V,L167M, G169G, E172Q, W201R, I219T, A221Y, D221N, G222A, I224T, G232C,N253D, G263R, G263W, E266K, V269F, L272V, L272P, G275R, G275R, E277K,V280G, D281N, F287V, F287L, V288L, D293N, D293V, A295I, A295V, I310N,I314T, E315K, N316K, V320L, V320M, S330R, D331N, D331G, D331E, G332S,V335M, A336S, R337W, R337P, R337Q, D339N, D339Q, G341A, G341D, I342F,K348N, A352D, I357T, G358R, G358E, R359C, R359H, C360Y, N361D, G364D,K365M, V368F, T371I, L374P, S376I, T384M, R385W, R385K, E387G, D390N,A392T, N393D, N393S, N393K, A394S, A394D, A394V, V395L, D397V, G398A,M403I, G406R, E407K, E407G, T408P, T408A, T408I, K410E, G411S, G411A,Q421K, A423A, A423A, R426W, R426Q, E427A, E427N, A431T, R449C, I457V,G458D, A459V, V460M, A468V, A468G, A470D, T477A, R479C, R479H, S485F,R486W, R486L, R488Q, R490W, I494T, A495T, A495V, R498C, R498H, A503V,R504L, Q505E, V506I, R510Q, G511R, G511E, R518S, R531C, R532W, R532Q,E538D, G540R, D550V, V552M, G557A, R559G, R559P, N566K, M568V, R569Q,R569L, Q58X, E174X, W201X, E241X, R270X, E440X, R486X, Q501X, L508X,R510X, E538X, R559X. These mutations are described in Canu et. al.,Blood Cells, Molecules and Diseases 2016, 57, pp. 100-109. In anembodiment, the mutant PKR is selected from G332S, G364D, T384M, K410E,R479H, R479K, R486W, R532W, R510Q, and R490W. In certain embodiments,the mutant PKR is selected from A468V, A495V, 190N, T408I, and Q421K,and R498H. In certain embodiments, the mutant PKR is R532W, K410E, orR510Q.

In one embodiment of the invention provided is a method of treatinganemia in a subject comprising administering to the subject an effectiveamount of (1) a compound described herein (e.g., a compound of Formulas(I)-(V), in the Examples, and in Table 1 and FIG. 1, or apharmaceutically acceptable salt thereof; (2) a pharmaceuticallyacceptable composition comprising a compound described herein or apharmaceutically acceptable salt thereof, and a pharmaceuticallyacceptable carrier. In certain embodiments, the anemia is adyserythropoietic anemia such as congenital dyserythropoietic anemiatype I, II, 11, or IV. In certain embodiments, the anemia is hemolyticanemia. In certain embodiments, the hemolytic anemia is a congenitaland/or hereditary form of hemolytic anemia such as PKD, sickle celldisease, thalassemias (e.g. alpha or beta), hereditary spherocytosis,hereditary elliptocytosis), paroxysmal nocturnal hemoglobinuria,abeta-liproteinemia (Bassen-Kornzweig syndrome). In certain embodiments,the hemolytic anemia is acquired hemolytic anemia such as autoimmunehemolytic anemia, drug-induced hemolytic anemia. In certain embodiments,the hemolytic anemia is anemia as part of a multi-system disease, suchas the anemia of Congenital Erythropoietic Purpura, Fanconi,Diamond-Blackfan.

As used herein, the term “anemia” refers to a deficiency of red bloodcells (RBCs) and/or hemoglobin. As used herein, anemia includes alltypes of clinical anemia, for example (but not limited to): microcyticanemia, iron deficiency anemia, hemoglobinopathies, heme synthesisdefect, globin synthesis defect, sideroblastic defect, normocyticanemia, anemia of chronic disease, aplastic anemia, hemolytic anemia,macrocytic anemia, megaloblastic anemia, pernicious anemia, dimorphicanemia, anemia of prematurity, Fanconi anemia, hereditary spherocytosis,sickle cell disease, warm autoimmune hemolytic anemia, cold agglutininhemolytic anemia, osteopetrosis, thalassemia, and myelodysplasticsyndrome.

In certain embodiments, anemia can be diagnosed on a complete bloodcount. In certain embodiments, anemia can be diagnosed based on themeasurement of one or more markers of hemolysis (e.g. RBC count,hemoglobin, reticulocytes, schistocytes, lactate Dehydrogenase (LDH),haptoglobin, bilirubin, and ferritin) and/or hemosiderinuria meancorpuscular volume (MCV) and/or red cell distribution width (RDW). Inthe context of the present invention, anemia is present if an individualhas a hemoglobin (Hb) less than the desired level, for example, the Hbconcentration of less than 14 g/dL, more preferably of less than 13g/dL, more preferably of less than 12 g/dL, more preferably of less than11 g/dL, or most preferably of less than 10 g/dL.

In certain embodiments, provided herein is a method of increasing amountof hemoglobin in a subject in thereof by administering an effectiveamount of a compound as described herein, or a pharmaceuticallyacceptable salt thereof, or a pharmaceutically acceptable compositionthereof. In certain embodiments, the provided method increaseshemoglobin concentration in the subject. In certain embodiments, theprovided method increases Hb concentration to a desired level, forexample, above 10 g/dL, more preferably above 11 g/dL, more preferablyabove 12 g/dL, more preferably above 13 g/dL, or most preferably above14 g/dL. In certain embodiments, the provided method increases Hbconcentration by at least about 0.5 g/dL. In certain embodiments, theprovided method increases Hb concentration by at least about 1.0 g/dL.In certain embodiments, the provided method increases Hb concentrationby at least about 1.5 g/dL. In certain embodiments, the provided methodincreases Hb concentration by at least about 2.0 g/dL. In certainembodiments, the provided method increases Hb concentration by at leastabout 2.5 g/dL. In certain embodiments, the provided method increases Hbconcentration by at least about 3.0 g/dL. In certain embodiments, theprovided method increases Hb concentration by at least about 3.5 g/dL.In certain embodiments, the provided method increases Hb concentrationby at least about 4.0 g/dL. In certain embodiments, the provided methodincreases Hb concentration by at least about 4.5 g/dL. In certainembodiments, the provided method increases Hb concentration by at leastabout 5.0 g/dL. In certain embodiments, the provided method increases Hbconcentration by at least about 5.5 g/dL. In certain embodiments, theprovided method increases Hb concentration by at least about 6.0 g/dL.

In one embodiment of the invention provided is a method for treatinghemolytic anemia comprising administering to a subject an effectiveamount of (1) a compound described herein (e.g., a compound of Formulas(I)-(V), in the Examples, and in Table 1 and FIG. 1, or apharmaceutically acceptable salt thereof; (2) a pharmaceuticallyacceptable composition comprising a compound described herein or apharmaceutically acceptable salt thereof, and a pharmaceuticallyacceptable carrier.

In a further embodiment, the hemolytic anemia is hereditary and/orcongenital hemolytic anemia, acquired hemolytic anemia, or anemia aspart of a multisystem disease. In certain embodiments, the hemolyticanemia is congenital anemia. In certain embodiments, the hemolyticanemia is hereditary (e.g. non-spherocytic hemolytic anemia orhereditary spherocytosis).

In one embodiment of the invention provided is a method of treatingthalassemia; hereditary spherocytosis; hereditary elliptocytosis;abetalipoproteinemia or Bassen-Kornzweig syndrome; paroxysmal nocturnalhemoglobinuria; acquired hemolytic anemia (e.g., congenital anemias(e.g., enzymopathies)); sickle cell disease; or anemia of chronicdiseases comprising administering to a subject an effective amount of(1) a compound described herein (e.g., a compound of Formulas (I)-(V),in the Examples, and in Table 1 and FIG. 1, or a pharmaceuticallyacceptable salt thereof; (2) a pharmaceutically acceptable compositioncomprising a compound described herein or a pharmaceutically acceptablesalt thereof, and a pharmaceutically acceptable carrier. In oneembodiment, the acquired hemolytic anemia comprises congenital anemias.In certain embodiments, the provided method is to treat thalassemia. Incertain embodiments, the thalassemia is beta-thalassemia.

As used herein, thalassemia is an inherited blood disorder in which thebody makes an abnormal form of hemoglobin. In certain embodiments, thedisorder results in large numbers of red blood cells being destroyed,which leads to anemia. In certain embodiments, the thalassemia is alphathalassemia. In certain embodiments, the thalassemia is betathalassemia.

In one embodiment of the invention provided is a method for activatingmutant PKR in red blood cells comprising administering to a subject inneed thereof an effective amount of (1) a compound described herein(e.g., a compound of Formulas (I)-(V), in the Examples, and in Table 1and FIG. 1, or a pharmaceutically acceptable salt thereof; (2) apharmaceutically acceptable composition comprising a compound describedherein or a pharmaceutically acceptable salt thereof, and apharmaceutically acceptable carrier. In one embodiment, the method is anex vive method. In another embodiment, the method is an in vitro method.In some embodiments, the blood or the red blood cells are derived orobtained from a subject suffering from or susceptible to a disease ordisorder selected from the group consisting of pyruvate kinasedeficiency (PKD), thalassemia (e.g., beta thalassemia), hereditaryspherocytosis, hereditary elliptocytosis, abetalipoproteinemia orBassen-Kornzweig syndrome, sickle cell disease, paroxysmal nocturnalhemoglobinuria, anemia (e.g., dyserythropoetic anemia), hemolyticanemia, and anemia of chronic diseases. In some embodiments, thehemolytic anemia is hereditary and/or congenital hemolytic anemia,acquired hemolytic anemia, or anemia as part of a multi-system disease.

In one embodiment of the invention provided is a method for activatingwild-type PKR in red blood cells comprising administering to a subjectin need thereof an effective amount of (1) a compound described herein(e.g., a compound of Formulas (I)-(V), in the Examples, and in Table 1and FIG. 1, or a pharmaceutically acceptable salt thereof; (2) apharmaceutically acceptable composition comprising a compound describedherein or a pharmaceutically acceptable salt thereof, and apharmaceutically acceptable carrier. In one embodiment, the method is anex vive method. In another embodiment, the method is an in vitro method.In some embodiments, the blood or the red blood cells are derived orobtained from a subject suffering from or susceptible to a disease ordisorder selected from the group consisting of pyruvate kinasedeficiency (PKD), thalassemia (e.g., beta thalassemia), hereditaryspherocytosis, hereditary elliptocytosis, abetalipoproteinemia orBassen-Kornzweig syndrome, sickle cell disease, paroxysmal nocturnalhemoglobinuria, anemia (e.g., dyserythropoetic anemia), hemolyticanemia, and anemia of chronic diseases. In some embodiments, thehemolytic anemia is hereditary and/or congenital hemolytic anemia,acquired hemolytic anemia, or anemia as part of a multi-system disease.

In one embodiment of the invention provided is a use of (1) a compounddescribed herein (e.g., a compound of Formulas (I)-(V), in the Examples,and in Table 1 and FIG. 1, or a pharmaceutically acceptable saltthereof; (2) a pharmaceutically acceptable composition comprising acompound described herein or a pharmaceutically acceptable salt thereof,and a pharmaceutically acceptable carrier for the preparation of amedicament for increasing the lifetime of red blood cells (RBCs) in needthereof.

In a further embodiment the compound or pharmaceutical composition isformulated to be added directly to whole blood or packed red blood cellsextracorporeally. In another embodiment, the compound or pharmaceuticalcomposition is formulated to be administered to a subject in needthereof.

In one embodiment of the invention provided is a use of (1) a compounddescribed herein (e.g., a compound of Formulas (I)-(V), in the Examples,and in Table 1 and FIG. 1, or a pharmaceutically acceptable saltthereof; (2) a pharmaceutically acceptable composition comprising acompound described herein or a pharmaceutically acceptable salt thereof,and a pharmaceutically acceptable carrier for the preparation of amedicament for regulating 2,3-diphosphoglycerate levels in blood in needthereof.

In one embodiment of the invention provided is a use of (1) a compounddescribed herein (e.g., a compound of Formulas (I)-(V), in the Examples,and in Table 1 and FIG. 1, or a pharmaceutically acceptable saltthereof; (2) a pharmaceutically acceptable composition comprising acompound described herein or a pharmaceutically acceptable salt thereof,and a pharmaceutically acceptable carrier for the preparation of amedicament for treating anemia. In certain embodiments, the anemia is adyserythropoietic anemia such as congenital dyserythropoietic anemiatype I, II, II, or IV. In certain embodiments, the anemia is hemolyticanemia. In certain embodiments, the hemolytic anemia is a congenitaland/or hereditary form of hemolytic anemia such as PKD, sickle celldisease, thalassemias (e.g. alpha or beta), hereditary spherocytosis,hereditary elliptocytosis), paroxysmal nocturnal hemoglobinuria,abeta-liproteinemia (Bassen-Kornzweig syndrome). In certain embodiments,the hemolytic anemia is acquired hemolytic anemia such as autoimmunehemolytic anemia, drug-induced hemolytic anemia. In certain embodiments,the hemolytic anemia is anemia as part of a multi-system disease, suchas the anemia of Congenital Erythropoietic Purpura, Fanconi,Diamond-Blackfan.

In one embodiment of the invention provided is a use of (1) a compounddescribed herein (e.g., a compound of Formulas (I)-(V), in the Examples,and in Table 1 and FIG. 1, or a pharmaceutically acceptable saltthereof; (2) a pharmaceutically acceptable composition comprising acompound described herein or a pharmaceutically acceptable salt thereof,and a pharmaceutically acceptable carrier for the preparation of amedicament for treating hemolytic anemia.

In one embodiment of the invention provided is a use of (1) a compounddescribed herein (e.g., a compound of Formulas (I)-(V), in the Examples,and in Table 1 and FIG. 1, or a pharmaceutically acceptable saltthereof; (2) a pharmaceutically acceptable composition comprising acompound described herein or a pharmaceutically acceptable salt thereof,and a pharmaceutically acceptable carrier for the preparation of amedicament for treating sickle cell disease.

In one embodiment of the invention provided is a use of (1) a compounddescribed herein (e.g., a compound of Formulas (I)-(V), in the Examples,and in Table 1 and FIG. 1, or a pharmaceutically acceptable saltthereof; (2) a pharmaceutically acceptable composition comprising acompound described herein or a pharmaceutically acceptable salt thereof,and a pharmaceutically acceptable carrier for the preparation of amedicament for treating pyruvate kinase deficiency (PKD) in a subject.

As described herein, PKD is a deficiency of PKR. In certain embodiments,the deficiency of PKR is associated with a PKR mutation.

In one embodiment of the invention provided is a use of (1) a compounddescribed herein (e.g., a compound of Formulas (I)-(V), in the Examples,and in Table 1 and FIG. 1, or a pharmaceutically acceptable saltthereof; (2) a pharmaceutically acceptable composition comprising acompound described herein or a pharmaceutically acceptable salt thereof,and a pharmaceutically acceptable carrier for the preparation of amedicament for treating thalassemia; hereditary spherocytosis;hereditary elliptocytosis; abetalipoproteinemia or Bassen-Kornzweigsyndrome; paroxysmal nocturnal hemoglobinuria; acquired hemolyticanemia; or anemia of chronic diseases.

In one embodiment of the invention provided is a use of (1) a compounddescribed herein (e.g., a compound of Formulas (I)-(V), in the Examples,and in Table 1 and FIG. 1, or a pharmaceutically acceptable saltthereof; (2) a pharmaceutically acceptable composition comprising acompound described herein or a pharmaceutically acceptable salt thereof,and a pharmaceutically acceptable carrier for the preparation of amedicament for activating mutant PKR in red blood cells.

In one embodiment of the invention provided is a use of (1) a compounddescribed herein (e.g., a compound of Formulas (I)-(V), in the Examples,and in Table 1 and FIG. 1), or a pharmaceutically acceptable saltthereof; (2) a pharmaceutically acceptable composition comprising acompound described herein or a pharmaceutically acceptable salt thereof,and a pharmaceutically acceptable carrier for the preparation of amedicament for activating wild-type PKR in red blood cells.

In one embodiment of the invention, provided is a method of activatingpyruvate kinase R (PKR), comprising contacting the PKR with an effectiveamount of of (1) a compound described herein (e.g., a compound ofFormulas (I)-(V), in the Examples, and in Table 1 and FIG. 1), or apharmaceutically acceptable salt thereof; or (2) a pharmaceuticallyacceptable composition comprising a compound described herein or apharmaceutically acceptable salt thereof, and a pharmaceuticallyacceptable carrier. In one embodiment, the PKR is a wild-type PKR. Inanother embodiment, the PKR is a mutant PKR. In some embodiments, thePKR is expressed in red blood cells. In one embodiment, the method is anex vivo method. In another embodiment, the method is an in vitro method.In some embodiments, the blood or the red blood cells are derived orobtained from a subject suffering from or susceptible to a disease ordisorder selected from the group consisting of pyruvate kinasedeficiency (PKD), thalassemia (e.g., beta thalassemia), hereditaryspherocytosis, hereditary elliptocytosis, abetalipoproteinemia orBassen-Kornzweig syndrome, sickle cell disease, paroxysmal nocturnalhemoglobinuria, anemia (e.g., dyserythropoetic anemia), hemolyticanemia, and anemia of chronic diseases. In some embodiments, thehemolytic anemia is hereditary and/or congenital hemolytic anemia,acquired hemolytic anemia, or anemia as part of a multi-system disease.

Since the compounds and compositions described herein act on the samebiological pathway and have the similar mode of action as the compoundsdescribed in WO2012/151451, the compounds and compositions presentedherein can activate the PKR mutants as described in WO2012/151451.

Compositions and Routes of Administration

The compositions delineated herein include the compounds delineatedherein (e.g., a compound described herein), as well as additionaltherapeutic agents if present, in amounts effective for achieving amodulation of disease or disease symptoms, including those describedherein.

The term “pharmaceutically acceptable carrier or adjuvant” refers to acarrier or adjuvant that may be administered to a patient, together witha compound provided herewith, and which does not destroy thepharmacological activity thereof and is nontoxic when administered indoses sufficient to deliver an effective amount of the compound.

Pharmaceutically acceptable carriers, adjuvants and vehicles that may beused in the pharmaceutical compositions provided herewith include, butare not limited to, ion exchangers, alumina, aluminum stearate,lecithin, self-emulsifying drug delivery systems (SEDDS) such asd-α-tocopherol polyethyleneglycol 1000 succinate, surfactants used inpharmaceutical dosage forms such as Tweens or other similar polymericdelivery matrices, serum proteins, such as human serum albumin, buffersubstances such as phosphates, glycine, sorbic acid, potassium sorbate,partial glyceride mixtures of saturated vegetable fatty acids, water,salts or electrolytes, such as protamine sulfate, disodium hydrogenphosphate, potassium hydrogen phosphate, sodium chloride, zinc salts,colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone,cellulose-based substances, polyethylene glycol, sodiumcarboxymethylcellulose, polyacrylates, waxes,polyethylene-polyoxypropylene-block polymers, polyethylene glycol andwool fat. Cyclodextrins such as α-, β-, and γ-cyclodextrin, orchemically modified derivatives such as hydroxyalkylcyclodextrins,including 2- and 3-hydroxypropyl-β-cyclodextrins, or other solubilizedderivatives may also be advantageously used to enhance delivery ofcompounds of the formulae described herein.

The pharmaceutical compositions provided herewith may be administeredorally, parenterally, by inhalation spray, topically, rectally, nasally,buccally, vaginally or via an implanted reservoir, preferably by oraladministration or administration by injection. The pharmaceuticalcompositions provided herewith may contain any conventional non-toxicpharmaceutically-acceptable carriers, adjuvants or vehicles.

The pharmaceutical compositions provided herewith may be orallyadministered in any orally acceptable dosage form including, but notlimited to, capsules, tablets, emulsions and aqueous suspensions,dispersions and solutions. In the case of tablets for oral use, carrierswhich are commonly used include lactose and corn starch. Lubricatingagents, such as magnesium stearate, are also typically added. For oraladministration in a capsule form, useful diluents include lactose anddried corn starch. When aqueous suspensions and/or emulsions areadministered orally, the active ingredient may be suspended or dissolvedin an oily phase is combined with emulsifying and/or suspending agents.

When the compositions provided herewith comprise a combination of acompound of the formulae described herein and one or more additionaltherapeutic or prophylactic agents, both the compound and the additionalagent should be present at dosage levels of between about 1 to 100%, andmore preferably between about 5 to 95% of the dosage normallyadministered in a monotherapy regimen. The additional agents may beadministered separately, as part of a multiple dose regimen, from thecompounds provided herewith. Alternatively, those agents may be part ofa single dosage form, mixed together with the compounds providedherewith in a single composition.

The compounds described herein can, for example, be administered byinjection, intravenously, intraarterially, subdermally,intraperitoneally, intramuscularly, or subcutaneously; or orally,buccally, nasally, transmucosally, topically, in an ophthalmicpreparation, or by inhalation, with a dosage ranging from about 0.5 toabout 100 mg/kg of body weight, alternatively dosages between 1 mg and1000 mg/dose, every 4 to 120 hours, or according to the requirements ofthe particular drug. The amount of active ingredient that may becombined with the carrier materials to produce a single dosage form willvary depending upon the host treated and the particular mode ofadministration. A typical preparation will contain from about 5% toabout 95% active compound (w/w). Alternatively, such preparationscontain from about 20% to about 80% active compound.

EXPERIMENTAL Abbreviations List

abbrv. Full Name abbrv. Full Name anhy. anhydrous aq. aqueous minminute(s) satd. saturated mL milliliter hrs hours mmol millimole(s) molmole(s) MS mass spectrometry NMR nuclear magnetic resonance TLC thinlayer chromatography HPLC high-performance liquid chromatography LCMSLiquid chromatography-mass MTBE Methyl tert-butyl ether spectrometryDAST Diethylaminosulfurtrifluoride CHCl₃ chloroform DCM dichloromethaneDMF dimethylformamide Et2O diethyl ether EtOH ethyl alcohol EtOAc ethylacetate MeOH methyl alcohol MeCN acetonitrile PE petroleum ether THFtetrahydrofuran DMSO dimethyl sulfoxide AcOH acetic acid HClhydrochloric acid H₂SO₄ sulfuric acid NH₄Cl ammonium chloride KOHpotassium hydroxide NaOH sodium hydroxide K₂CO₃ potassium carbonateNa₂CO₃ sodium carbonate TFA trifluoroacetic acid Na₂SO₄ sodium sulfateNaBH₄ sodium borohydride NaHCO₃ sodium bicarbonate LiHMDS lithium NaBH₄sodium borohydride hexamethyldisilylamide Et₃N or Triethylamine Py orPyr pyridine TEA DMAP 4-(dimethylamino)pyridine DIPEAN,N-diisopropylethylamine BINAP 2,2′bis(diphenylphosphanyl)- dppf 1,1′-1,1′-binaphthyl bis(diphenylphosphino)ferrocene PEP Phospho(enol)pyruvicacid LDH Lactate Dehydrogenase DTT DL-Dithiothreitol BSA Bovine serumAlbumin NADH β-Nicotinamide adenine SEM 2-(Trimethylsilyl)ethoxymethyldinucleotide, reduced p-TsOH p-Toluenesulfonic acid DCE1,2-dichloroethane

General Experimental

In the following examples, the chemical reagents were purchased fromcommercial sources (such as Alfa, Acros, Sigma Aldrich, TCI and ShanghaiChemical Reagent Company), and used without further purification. Flashchromatography was performed on an Ez Purifier II via column with silicagel particles of 200-300 esh. Analytical and preparative thin layerchromatography plates (TLC) were HSGF 254 (0.15-0.2 mm thickness,Shanghai Anbang Company, China). Nuclear magnetic resonance (NMR)spectra were recorded using Brucker AMX-300 or AMX-400 NMR (Brucker,Switzerland). Chemical shifts were reported in parts per million (ppm,8) etero(ESI) from a Waters LCT TOF Mass Spectrometer (Waters, USA).HPLC chromatographs were recorded on Agilent 1200 Liquid Chromatography(Agilent, USA, column: Ultimate 4.6 m×50 mm, 5 M, mobile phase A: 0.1%formic acid in water; mobile phase B: acetonitrile). Microwave reactionswere run on an Initiator 2.5 Microwave Synthesizer (Biotage, Sweden).

HPLC conditions used in the experiments described herein are as follows:

Method 1: Instrument: Shimadzu LC-2010AHT Column: YMC-Triart C18, 50×4.6mm, 5 μm

Mobilephase: Solvent A: H₂O/CH₃OH/TFA=90/10/0.1,

-   -   Solvent B: H₂O/CH₃OH/TFA=90/10/0.1        Flow rate: 2.5 mL/min; Column temperature: 35° C.; Wavelength:        220 nm/254 nm

Method 2: Instrument: Shimadzu LC-2010AHT Column: YMC-Triart C18, 50×4.6mm, 5 μm

Mobilephase: Solvent A: H₂O/CH₃OH/TFA=90/10/0.1,

-   -   Solvent B: H₂O/CH₃OH/TFA=90/10/0.1        Flow rate: 2.5 mL/min; Column temperature: 35° C.; Wavelength:        220 nm/254 nm

Prep-HPLC conditions used in the experiments described herein are asfollows:

Instrument: Waters 2545B/2767 Column: YMC-Triart C18, 50×4.6 mm, 5 μmMobilephase: Solvent A: H₂O (01.% FA),

-   -   Solvent B: CH₃OH or CH₃CN        Flow rate: 20 mL/min; Column temperature: 35° C.; Wavelength:        220 nm/254 nm

Example 1. Synthesis of6-(3-methoxybenzyl)-2,4-dimethyl-4,6-dihydro-5H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazin-5-one

Step A. Ethyl (Z)-2-azido-3-(2-methylthiazol-5-yl)acrylate

To a solution of NaOEt (803 mg, 11.79 mmol) in EtOH (10 mL) betweenabout −10° C. and about −5° C. was added drop wise a solution of2-methylthiazole-5-carbaldehyde (500 mg, 3.93 mmol) and ethyl2-azidoacetate (1.53 g, 11.79 mmol) in anhydrous EtOH (3 mL). Thereaction mixture was stirred for about 1 hr. while the temperaturemaintained below 0° C., then warmed to r.t. and stirred for another 2hr. The resulting mixture was poured into saturated aqueous NH₄Cl (50mL) at 0° C. and extracted with EtOAc. The combined organic layers werewashed with brine, dried over anhydrous Na₂SO₄ and concentrated underreduced pressure to give the desired product (500 mg) which was directlyused in the next step without any purification. LCMS: m/z 239 (M+H)⁺.

Step B. Ethyl 2-methyl-4H-pyrrolo[2,3-d]thiazole-5-carboxylate

A mixture of ethyl (Z)-2-azido-3-(2-methylthiazol-5-yl)acrylate (500 mg,2.1 mmol) in o-xylene (5 mL) was stirred at 140° C. for 2 hr. thencooled down to r.t. and then directly purified by column chromatographyon silica gel (eluent: pentane/EtOAc=6/1 to give the desired product(220 mg, 49.8% yield). LCMS: m/z 211 (M+H)⁺.

Step C. Ethyl 2,4-dimethyl-4H-pyrrolo[2,3-d]thiazole-5-carboxylate

To a solution of ethyl 2-methyl-4H-pyrrolo[2,3-d]thiazole-5-carboxylate(160 mg, 0.76 mmol) in DMF (3 mL) at 0° C. was added NaH (36.5 mg, 1.52mmol). The reaction mixture was stirred at r.t. for 0.5 hr., followed byaddition of CH₃I (47 μL, 0.76 mmol). The resulting mixture was stirredat r.t for 0.5 hr. then poured into saturated aqueous NH₄Cl at 0° C. andextracted with EtOAc. The combined organic layers were washed withbrine, dried over anhydrous Na₂SO₄ and concentrated under reducedpressure. The residue was purified by column chromatography on silicagel (eluent: pentane/ethyl acetate=6/1) to give the desired product (124mg, 72.6% yield). LCMS: m/z 225 (M+H)⁺.

Step D. Ethyl6-formyl-2,4-dimethyl-4H-pyrrolo[2,3-d]thiazole-5-carboxylate

To a mixture of ethyl2,4-dimethyl-4H-pyrrolo[2,3-d]thiazole-5-carboxylate (100 mg, 0.446mmol) in DMF (1 mL)) at 0° C. was added POCl₃(122.5 μL, 1.338 mmol). Thereaction mixture was stirred at 100° C. for 2 hr. then poured intosaturated aqueous NaHCO₃ at 0° C. and extracted with EtOAc. The combinedorganic layers were washed with brine, dried over anhydrous Na₂SO₄ andconcentrated under reduced pressure. The residue was purified by columnchromatography on silica gel (eluent: pentane/ethyl acetate=5/1) to givethe desired product (57 mg, 50.7% yield). LCMS: m/z 253 (M+H)⁺.

Step E.2,4-Dimethyl-4,6-dihydro-5H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazin-5-one

To a mixture of ethyl6-formyl-2,4-dimethyl-4H-pyrrolo[2,3-d]thiazole-5-carboxylate (57 mg,0.226 mmol) in 2-ethoxyethanol (2 mL) was added N₂H₄.H₂O (53.7 μL, 1.130mmol). The reaction mixture was stirred at 100° C. for 1 hr. then pouredinto H₂O and extracted with EtOAc. The combined organic layers werewashed with brine; dried over anhydrous Na₂SO₄ and concentrated underreduced pressure. The residue was purified by column chromatography onsilica gel (eluent: pentane/ethyl acetate=5/1) to give the desiredproduct (49 mg, 98.4% yield). LCMS: m/z 221 (M+H)⁺.

Step F.6-(3-Methoxybenzyl)-2,4-dimethyl-4,6-dihydro-5H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazin-5-one

To a mixture of2,4-dimethyl-4,6-dihydro-5H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazin-5-one(49 mg, 0.223 mmol) in DMF (1 mL) at 0° C. was added t-BuOK (50.8 mg,0.454 mmol). The reaction mixture was stirred at r.t. for 0.5 hr.,followed by addition of 1-(chloromethyl)-3-methoxybenzene (34.9 mg,0.223 mmol). The resulting mixture was stirred at r.t. for 1 hr. thenpoured into saturated aqueous NH₄Cl solution at 0° C. and extracted withEtOAc. The combined organic layers were washed with brine, dried overanhydrous Na₂SO₄ and concentrated under reduced pressure. The residuewas purified by column chromatography on silica gel (eluent:pentane/ethyl acetate=3/1) to give the desired product. LCMS: m/z 341(M+H)⁺. ¹H NMR (400 MHz, DMSO-d₆) δ 8.56 (s, 1H), 7.23 (t, 1H),6.92-6.72 (m, 3H), 5.32 (s, 2H), 4.26 (s, 3H), 3.72 (s, 3H), 2.85 (s,3H).

Example 1 provides exemplified synthesis of compound E1-3. It isunderstood that different compounds can be synthesized with appropriateheteroaryl-CH₂-halide. Examples 2. Preparation of Compounds of FormulaE2-vii with Scheme E2

Wherein X is a leaving group (e.g. Cl, Br or I, OMs, or OTs); M is anorganic metal complex (e.g. organoboron complex such as boronic acid orpinaco boron complex, organotin complex such as —Sn(Bu^(i))₃; organozinccomplex such as —Zn(halogen)); Q and R² are each independentlyoptionally substituted 5-membered or 6-membered heteroaryl. Similar tothe synthesis of compounds of Formula E1-v in Example 1, compound E2-ivcan be synthesized from thiazole aldehyde E2-i with a few modifications(e.g. reaction of compound E2-ii with MsCl followed by elimination togive compound E2-iii; the tricyclic system can be formed with cat. AcOHin 2-methoxyethanol). Substitution and iodination of compound E2-ivprovides compound E2-vi. Coupling of compound E2-vi with organometal inpresence of a catalyst gives compound E2-vii.

Example 2A. Synthesis of4-methyl-4,6-dihydro-5H-thiazolo[5′,4′:4,5]pyrrolo-[2,3-d]pyridazin-5-one

Step A. Ethyl 2-azido-3-hydroxy-3-(thiazol-5-yl)propanoate

Sodium (12.2 g, 0.531 mol) was slowly added at r.t. to a stirredsolution of dry EtOH (300 mL). The reaction mixture was then cooled to−20° C., followed by drop wise addition of a solution of ethyl2-azidoacetate (68.5 g, 0.531 mol) and thiazole-5-carbaldehyde (20.0 g,0.177 mol) in anhydrous EtOH (100 mL) while keeping the temperaturebetween −20° C. to −15° C. After the addition, the reaction mixture wasstirred at −20° C. for additional 1 hr. and then poured into saturatedaqueous NH₄Cl (1L). The resulting mixture was saturated with NaCl andextracted with EtOAc. The combined organic phase was washed with brine,dried over anhydrous Na₂SO₄ and concentrated under reduced pressure. Theresidue was purified by column chromatography on silica gel using(eluent: PE/EtOAc=6/1 to 5/1 to 1/1) to afford desired product (34 g) aspale. LCMS: m/z=243 (M+H)⁺.

Step B. Ethyl (Z)-2-azido-3-(thiazol-5-yl)acrylate

To a stirred mixture of ethyl2-azido-3-hydroxy-3-(thiazol-5-yl)propanoate (103 g, 0.426 mol) in dryDCM (1.5 L) at −35° C. was added MsCl (146 g, 1.28 mol), followed bydrop wise addition of TEA (301 g, 2.98 mol) while keeping thetemperature between −35° C. to −30° C. After the addition, the reactionmixture was stirred at −30° C. for another 15 min then poured intosaturated aqueous NH₄Cl (1.5 L). The resulting mixture was saturatedwith NaCl and extracted with DCM twice. The combined organic layers werewashed in sequence with aqueous HCl (1 M) and brine, dried overanhydrous Na₂SO₄ and concentrated under reduced pressure. The residuewas purified by column chromatography on silica gel using (eluent:PE/EtOAc=5/1) to afford the desired product (82.0 g, 86.3% yield). LCMS:m/z=225 (M+H)⁺.

Steps C-E to synthesize ethyl 4H-pyrrolo[2,3-d]thiazole-5-carboxylate,ethyl 4-methyl-4H-pyrrolo[2,3-d]thiazole-5-carboxylate, and ethyl6-formyl-4-methyl-4H-pyrrolo[2,3-d]thiazole-5-carboxylate were similarto the procedures in Example 1.

Step F. Ethyl(E)-6-(hydrazonomethyl)-4-methyl-4H-pyrrolo[2,3-d]thiazole-5-carboxylate

To a stirred mixture of N₂H₄.H₂O (2.0 g, 98%, 40 mmol) in2-methoxyethanol (50 mL) at r.t. was added ethyl6-formyl-4-methyl-4H-pyrrolo[2,3-d]thiazole-5-carboxylate (4.8 g, 20mmol), followed by addition of 20 drops of AcOH. The reaction mixturewas stirred at r.t. for about 30 min till the mixture turned clear. Theresulting mixture was poured into water (100 mL) with stirring andextracted with DCM. The combined organic layers were dried overanhydrous Na₂SO₄ and concentrated under reduced pressure to afford thedesired product which was used in the next step without furtherpurification. LCMS: m/z=253 (M+H)⁺.

Step G.4-Methyl-4,6-dihydro-5H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazin-5-one

To a stirred suspension of ethyl(E)-6-(hydrazonomethyl)-4-methyl-4H-pyrrolo[2,3-d]thiazole-5-carboxylate(4.8 g, 0.19 mol) in 2-methoxyethanol (50 mL) at r.t. was added AcOH (20drops). The reaction suspension was stirred at 105° C. for 3 hr. andthen filtered. The filter cake was washed with water and dried underhigh vacuum to get the first batch of the desired product. The filtratewas diluted with water and extracted with DCM twice. The organic layerswere washed with brine, dried over anhydrous Na₂SO₄ and concentratedunder reduced pressure to afford the second batch of the desiredproduct. The combined two batches of the desired product (2.5 g) wasdirectly used in the next step without further purification. LCMS:m/z=207 (M+H)⁺. 1H NMR (400 MHz, DMSO) δ 12.68 (s, 1H), 9.35 (s, 1H),8.55 (s, 1H), 4.30 (s, 3H)

Example 3. Synthesis of Compounds E3-vii and E3-viii

wherein Hal is halogen (e.g. Cl, Br or I); LG is a leaving group (e.g.Cl, Br, I, OMs, or OTs); Q is optionally substituted 5-membered or6-membered heteroaryl; and Q′ is further functionalized i-membered or6-membered heteroaryl. Aromatic substitution reaction of compound E3-iwith sodium methanethiolate provides compound E3-ii, which can beconverted to compound E3-v using the synthesis of compound E1-iii toE1-vi. Oxidation of compound E3-v with mCPBA gives compound E3-vi andE3-vii respectively.

Example 3A. Synthesis of4-methyl-2-(methylthio)-4,6-dihydro-5H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazin-5-one

Step A: Ethyl4-methyl-2-(methylthio)-4H-pyrrolo[2,3-d]thiazole-5-carboxylate

To a mixture of ethyl2-bromo-4-methyl-4H-pyrrolo[2,3-d]thiazole-5-carboxylate (500.0 mg, 1.73mmol) in EtOH (10.0 mL) was added NaSMe (240.0 mg, 3.5 mmol). Thereaction mixture was stirred at 25° C. for 3 hr then quenched with icewater and extracted with DCM.

The combined organic layers were washed with brine, dried over anhydrousNa₂SO₄ and concentrated under reduced pressure to afford desired product(460 mg) which was directly used in the next step without anypurification. LC-MS: m/z 257 (M+H)⁺.

Step B: Ethyl6-formyl-4-methyl-2-(methylthio)-4H-pyrrolo[2,3-d]thiazole-5-carboxylate

To a solution of ethyl4-methyl-2-(methylthio)-4H-pyrrolo[2,3-d]thiazole-5-carboxylate (460.0mg, 1.8 mmol) and N-methyl-N-phenylformamide (490 mg, 3.6 mmol) in DCE(10 mL) was added POCl₃(550.0 mg, 3.6 mmol). The resulting mixture wasstirred at 130° C. for 3 hr. then quenched with ice water and extractedwith DCM. The combined organic layers were washed with brine, dried overanhydrous Na₂SO₄ and concentrated under reduced pressure. The residuewas purified by column chromatography on silica gel (eluent:PE/EtOAc=8/1) to give the desired product (320.0 mg, 63% yield). LC-MS:m/z 285 (M+H)⁺.

Step C:4-Methyl-2-(methylthio)-4,6-dihydro-5H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazin-5-one

To a solution of ethyl6-formyl-4-methyl-2-(methylthio)-4H-pyrrolo[2,3-d]thiazole-5-carboxylate(300.0 mg, 1.06 mmol) in EtOH (5.0 mL) was added N₂H₄.H₂O (2 mL, 98%wt). The reaction mixture was stirred at r.t. for 1 hr. then heated to60° C. for overnight then cooled down. The solid was collected byfiltration and dried under high vacuum to afford the desired product(180.0 mg, 67% yield). LC-MS: m/z 253 (M+H)⁺. ¹H NMR (400 MHz, DMSO-d₆)δ 12.61 (s, 1H), 8.48 (s, 1H), 4.22 (s, 3H), 2.81 (s, 3H).

Example 3B. Synthesis of6-((2-aminopyridin-4-yl)methyl)-4-methyl-2-(methylsulfinyl)-4H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one

Step A. 2-Bromo-4-(bromomethyl)pyridine

A mixture of 2-bromo-4-methylpyridine (1 g, 5.81 mmol), NBS (1.1 g, 6.39mmol) and a catalytic amount of AIBN (100 mg) in CCl₄ (10 mL) wasstirred at 80° C. overnight. The resulting mixture was concentratedunder reduced pressure and the residue was purified by columnchromatography on silica gel (eluent: PE/EtOAc=200/1) to give thedesired product E3-4 (500 mg, 34.28% yield).

Step B.6-((2-Bromopyridin-4-yl)methyl)-4-methyl-2-(methylthio)-4H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one

A mixture of4-methyl-2-(methylthio)-4H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one(100 mg, 0.40 mmol), and K₂CO₃ (164 mg, 1.19 mmol) in DMF (8 mL) wasstirred at 60° C. for 2 hr., followed by addition of a solution of2-bromo-4-(bromomethyl)pyridine (199 mg, 0.80 mmol) in DMF (2 mL) and acatalytic amount of TBAB (13 mg). The mixture was stirred at 60° C.overnight then quenched with water (20 mL) and extracted with EtOAc. Thecombined organic layers were washed with saturated aqueous NH₄Cl, driedover anhydrous Na₂SO₄ and concentrated under reduced pressure. Theresidue was purified by column chromatography on silica gel (eluent:PE/EtOAc=10/1) to give the desired product (150 mg, 89.62% yield). LCMS:m/z 423 (M+H)⁺.

Step C. Tert-butyl(4-((4-methyl-2-(methylthio)-5-oxo-4H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazin-6(5H)-yl)methyl)pyridin-2-yl)carbamate

A mixture of6-((2-bromopyridin-4-yl)methyl)-4-methyl-2-(methylthio)-4H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one(100 mg, 0.24 mmol), tert-butyl carbamate (83 mg, 0.71 mmol), K₃PO₄ (201mg, 0.95 mmol), Pd₂(dba)₃ (18 mg, 0.02 mmol) and Xantphos (11 mg, 0.02mmol) in dioxane (10 mL) was stirred at 100° C. under nitrogenovernight. The resulting mixture was quenched with water and extractedwith EtOAc. The combined organic layers were washed with brine, driedover anhydrous Na₂SO₄ and concentrated under reduced pressure. Theresidue was purified by column chromatography on silica gel (eluent:PE/EtOAc=3/1) to give the desired product (100 mg, 92.10% yield). LCMS:m/z 459 (M+H)⁺.

Step D. Tert-butyl(4-((4-methyl-2-(methylsulfinyl)-5-oxo-4H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazin-6(5H)-yl)methyl)pyridin-2-yl)carbamate

To a mixture oftert-butyl(4-((4-methyl-2-(methylthio)-5-oxo-4H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazin-6(5H)-yl)methyl)pyridin-2-yl)carbamate(100 mg, 0.22 mmol) in DCM (5 mL) at 0° C. was added3-chloroperoxybenzoic acid (38 mg, 0.22 mmol). The reaction mixture wasstirred at 0° C. for 1 hr. then quenched with water and extracted withDCM. The combined organic layers were washed with saturated aqueousNaHCO₃, dried over anhydrous Na₂SO₄ and concentrated under reducedpressure to give the desired product (100 mg, 96.63% yield) which wasdirectly used in the next step without any purification. LCMS: m/z 475(M+H)⁺.

Step E.6-((2-Aminopyridin-4-yl)methyl)-4-methyl-2-(methylsulfinyl)-4H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one

A mixture of tert-butyl(4-((4-methyl-2-(methylsulfinyl)-5-oxo-4H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazin-6(5H)-yl)methyl)pyridin-2-yl)carbamate(100 mg, 0.21 mmol) in TFA (1 mL) and DCM (3 mL) was stirred at r.t. for1 h then concentrated under reduced pressure. The residue was purifiedby prep-HPLC to give the desired product (20 mg, 25.35% yield). LCMS:m/z 375 (M+H)⁺. ¹H NMR (400 MHz, DMSO-d₆) δ 8.67 (s, 1H), 7.73 (d, 1H),6.30 (d, 1H), 6.12 (s, 1H), 5.89 (s, 2H), 5.24-5.03 (m, 2H), 4.29 (s,3H), 3.03 (s, 3H).

The procedure set forth above was used to produce following compoundsusing appropriate starting materials. Standard protection anddeprotection can be used when necessary.

Cpd No. Structure and chemical name Charaterization E3-8

LC-MS: m/z 376 (M + H)⁺. ¹H NMR (400 MHz, DMSO-d₆) δ 8.76 (s, 1H), 8.12(d, 1H), 6.60 (s, 2H), 6.22 (d, 1H), 5.20 (t, 2H), 4.28 (s, 3H), 3.12(s, 3H). E3-9

LC-MS: 376 (M + H)⁺. ¹H NMR (400 MHz, DMSO-d₆) δ 8.70 (s, 1H), 8.29 (s,2H), 6.66 (s, 2H), 5.16 (s, 2H), 4.28 (s, 3H), 3.10 (s, 3H).

Example 3C. Synthesis of6-((2-aminothiazol-5-yl)methyl)-4-methyl-2-(methylsulfinyl)-4H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one

Step A-B

2-Bromo-5-(bromomethyl)thiazole and6-((2-Bromothiazol-5-yl)methyl)-4-methyl-2-(methylthio)-4H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazin-5(6H)-onewere synthesized similar to Example 3B.

Step C.6-((2-((2,4-dimethoxybenzyl)amino)thiayl)-4-methyl-2-(methylthio)-4H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one

A mixture of6-((2-bromothiazol-5-yl)methyl)-4-methyl-2-(methylthio)-4H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one(130 mg, 0.30 mmol) and DIPEA (0.1 mL) in NMP (0.1 mL) and(2,4-dimethoxyphenyl)methanamine (0.1 mL) was stirred at 150° C. for 4hr. Then the traction mixture was quenched with water (10 mL), extractedwith EtOAc. The combined organic layers were washed with brine, driedover anhydrous Na₂SO₄ and concentrated under reduced pressure. Theresidue was purified by column chromatography on silica gel (eluted withPE/EtOAc=5/1) to give the desired product (60 mg, 38.4% yield). LC-MS:m/z 515 (M+H)⁺.

Step D.6-((2-((2,4-dimethoxybenzyl)amino)thiazol-5-yl)methyl)-4-methyl-2-(methylsulfinyl)-4H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one

To a mixture of6-((2-((2,4-dimethoxybenzyl)amino)thiazol-5-yl)methyl)-4-methyl-2-(methylthio)-4H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one(50 mg, 0.10 mmol) in THF (3 mL) at 0° C. was added oxone (61 mg, 0.10mmol). The mixture was stirred at 0° C. for 1 hr, then quenched withsaturated aqueous Na₂S₂O₃ solution (5 mL) and extracted with DCM. Thecombined organic layers were washed with brine, dried over anhydrousNa₂SO₄ and concentrated under reduced pressure to give the desiredproduct (30 mg, 50.1% yield) which was used directly in the next stepwithout further purification. LC-MS: m/z 531 (M+H)⁺.

Step E

6-((2-Aminothiazol-5-yl)methyl)-4-methyl-2-(methylsulfinyl)-4H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazin-5(6H)-onewas synthesized similar to Example 3B. LC-MS: m/z 381 (M+H)⁺. ¹H NMR(400 MHz, DMSO-d₆) δ 8.70 (s, 1H), 6.97 (s, 1H), 6.87 (s, 2H), 5.28 (s,2H), 4.29 (s, 3H), 3.11 (s, 3H).

The procedure set forth above was used to produce following compoundsusing appropriate starting materials. Standard protection anddeprotection can be used when necessary.

Cpd No. Structure and chemical name Charaterization E3-15

LCMS: m/z 381 (M + H)⁺. ¹H NMR (400 MHz, DMSO-d₆) δ 8.69 (s, 1H), 6.92(s, 2H), 6.21 (s, 1H), 5.26-5.05 (m, 2H), 4.30 (s, 3H), 3.11 (s, 3H).

Examples 4. Synthesis of6-((1H-pyrazol-3-yl)methyl)-2-((1H-pyrazol-5-yl)methyl)-4-methyl-4H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazin-(6H)-oneExample 4A. Synthesis of3-((phenylsulfonyl)methyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1II-pyrazole

Step A. Methyl1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazole-3-carboxylate

At 0° C. under N₂ atmosphere, to a stirred solution of methyl1H-pyrazole-3-carboxylate (90 g, 0.72 mol) in THF (1 L) was added NaH(20.7 g, 0.864 mol, 60%). The resulting mixture was slowly warmed up tor.t and stirred for 1 h. The reaction mixture was then cooled back to 0°C. and SEMCl (151.5 mL, 0.842 mol) was added drop wise. The stirring wascontinued for another 2 hr before quenched with sat NH₄Cl and extractedwith ethyl acetate (3×). The combined organic layers were washed withbrine and dried over Na₂SO₄. Solvents were removed under vacuum toprovide crude product 210 g which was used in the next step withoutpurification.

Step B. (1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-3-yl)methanol

At 0° C. under N₂ atmosphere, to the suspension of LAH (16.9 g, 0.44mol) in THF (760 mL) was added the crude methyl1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazole-3-carboxylate (76 g).The resulting mixture was slowly warmed up to r.t. and stirred for 1 hr.The reaction mixture was cooled back to 0° C. and H₂O (15.6 mL), 10%NaOH (15.6 mL), H₂O (15.6 mL) was added successively. The resultingmixture was filtered through a pad of celite and washed with MTBE (4×).The combined organic fractions were dried over Na₂SO₄. Solvents wereremoved under reduced pressure to provide crude product 69.4 g which wasused in the next step without purification. LC-MS: m/z 229 (M+H)⁺.

Step C. 3-(iodomethyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazole

At 0° C. under N₂ atmosphere, to a stirred solution of(1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-3-yl)methanol (61.5 g,theoretically 0.262 mol) in THF (310 mL) was added TEA (55.42 mL, 0.393mol) followed by MsCl (24 mL, 0.314 mol). The reaction was warmed up tor.t and stirred for 1 hr before the introduction of NaI (196.5 g, 1.31mol, in 310 mL DMF). The resulting mixture was stirred for another 1 hrand quenched with ice-water, extracted with MTBE (3×). The combinedorganic layers were washed with sat. Na₂S₂O₃ and brine, dried overNa₂SO₄ and concentrated to provide 77.5 g crude product used in the nextstep without purification. LC-MS: m/z 339 (M+H)⁺.

Step D.3-((phenylsulfonyl)methyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazole

At 0° C. under N₂ atmosphere, to a stirred solution of(1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-3-yl)methanol (77.5 g,theoretically 0.229 mol) in DMF (600 mL) was added sodiumbenzenesulfinate (53.5 g, 0.32 mol) and stirred for 1 hr at 0° C. Afterwarmed up to r.t., the reaction mixture was quenched with ice-water andsat. Na₂S₂O₃, extracted with ethyl acetate (3×). The combined organiclayers were washed with sat. NaHCO₃ and brined successively, dried overNa₂SO₄. Solvents were removed under vacuum and the residue was purifiedby flash chromatography (silica gel, 20%˜70% ethyl acetate in petroleumether) to provide 56.7 g as a light yellow oil. LCMS: [M+H]⁺ 353. 1H NMR(400 MHz, DMSO) δ 7.85-7.77 (m, 4H), 7.62 (dd, 2H), 6.19 (d, 1H), 5.35(d, 2H), 4.70 (d, 2H), 3.44-3.38 (m, 2H), 0.88-0.77 (m, 2H), −0.01 (s,9H).

Example 4B. Synthesis of6-((1H-pyrazol-3-yl)methyl)-2-((1H-pyrazol-5-yl)methyl)-4-methyl-4H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one

Step A.4-methyl-2-((phenylsulfonyl)(1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-5-yl)methyl)-4H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one

To a solution of3-((phenylsulfonyl)methyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazole(1.8 g, 5.1 mmol) in dry THP (30 mL) at −40° C. was added LiHMDS (7.5mL, 7.5 mmol) drop-wise. The mixture was stirred at room temperature for30 min, followed by addition of a suspension of4-methyl-2-(methylsulfinyl)-4H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one(580 mg, 2.7 mmol) in dry THF (30 mL) at room temperature. The mixturewas stirred at r.t. for another 1 hr and poured into ice-cooledsaturated aqueous NH₄Cl (20 mL) and extracted with EtOAc (3×100 mL). Thecombined organic layers were washed with water (60 mL), dried overanhydrous Na₂SO₄ and concentrated under reduced pressure. The residuewas purified by flash chromatography (silica gel, 0˜2.5% methanol indichloromethane) to give the desired product (800 mg, 66%). LC-MS (ESI)found: 557 (M+H)⁺. 1H NMR (400 MHz, DMSO) δ 12.78 (s, 1H), 8.65 (s, 1H),8.03 (d, 1H), 7.84-7.78 (m, 3H), 7.67-7.59 (m, 2H), 6.94 (s, 1H), 6.72(d, 1H), 5.48 (d, 2H), 4.29 (s, 3H), 3.56 (dd, 2H), 0.88 (dd, 2H), 0.00(s, 9H).

Step B.4-methyl-2-((1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-5-yl)methyl)-4H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one

To a mixture of4-methyl-2-((phenylsulfonyl)(1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-5-yl)methyl)-4H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one(0.8 g, 1.41 mmol) in THF (5 mL) and MeOH (10 mL) under N₂ was addeddropwise SmI₂ (0.1M/THF, 45 mL) under ice-bath After stirred for 10 min,the reaction was quenched with saturated aqueous NH₄Cl (50 mL) andextracted with EAOAc (50 mL×3). The combined organic layers were washedwith water (60 mL), dried over anhydrous Na₂SO₄ and concentrated underreduced pressure. The residue was purified by flash chromatography(silica gel, 0˜3% methanol in dichloromethane) to give the desiredproduct (310 mg, 51.0%). LC-MS found: 417 (M+H)⁺. ¹H NMR (400 MHz,CDCl₃) δ 8.31 (s, 1H), 7.60 (d, 1H), 6.39 (d, 1H), 5.49 (s, 2H), 4.58(s, 2H), 4.43 (s, 3H), 3.62 (t, 2H), 0.95 (t, 2H), 0.0 (s, 9H).

Step C. tert-butyl3-((4-methyl-5-oxo-2-((1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-5-yl)methyl)-4H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazin-6(5H)-yl)methyl)-1H-pyrazole-1-carboxylate

To a mixture of4-methyl-2-((1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-5-yl)methyl)-4H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one(30 mg, 0.07 mmol) and K₂CO₃ (25 mg, 0.18 mmol) in DMF (5.0 mL) wasbubbled with argon and stirred at 50° C. for 1 hr. Then tert-butyl3-(bromomethyl)-1H-pyrazole-1-carboxylate (37 mg, 0.14 mmol) was added.After stirred at 50° C. overnight under argon, the reaction mixture wascooled down to r.t. and quenched with saturated aqueous NH₄Cl (20 mL)and extracted with EAOAc (30 mL×3). The combined organic layers werewashed with water (40 mL), dried over anhydrous Na₂SO₄ and concentratedunder reduced pressure. The residue was purified by Prep-TLC(DCM:MeOH=15:1) to give desire product (15 mg, 35.0%). LC-MS (ESI)found: 597 (M+H)⁺.

Step D.6-((1H-pyrazol-3-yl)methyl)-2-((1H-pyrazol-5-yl)methyl)-4-methyl-4H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one

To a solution of tert-butyl3-((4-methyl-5-oxo-2-((1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-5-yl)methyl)-4H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazin-6(5H)-yl)methyl)-1H-pyrazole-1-carboxylate(15 mg, 0.025 mmol) in DCM (5 mL) was added TFA (1 mL). The reactionmixture was stirred at room temperature overnight before concentratedand purified by Pre-TLC (DCM:MeOH=15:1) to give desire product (5 mg,50%). LC-MS, found: 367 (M+H)⁺. ¹H NMR (400 MHz, DMSO) δ 12.80 (s, 1H),12.65 (s, 1H), 8.50 (s, 1H), 7.71 (brs, 1H), 7.60 (brs, 1H), 6.26 (d,1H), 6.11 (d, 1H), 5.32 (s, 2H), 4.49 (s, 2H), 4.27 (s, 3H).

Example 5. Synthesis of4-methyl-2-(methylsulfinyl)-4,6-dihydro-5H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazin-5-one

To a stirred suspension of4-methyl-2-(methylthio)-4,6-dihydro-5H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazin-5-one(1.01 g, 4.0 mmol) in DCM (20 mL) was added 3-chloro-benzoperoxoic acid(0.77 g, 3.8 mmol) at r.t. The mixture stirred at r.t. for 2 hr. Thenthe mixture was filtered washed with EtOAc and triturated with MeOH. Thesolid was dried in vacuum to give4-methyl-2-(methylsulfinyl)-4,6-dihydro-5H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazin-5-one(600 mg). LCMS: m/z 269 (M+H)⁺. 1H NMR (400 MHz, DMSO) δ 12.78 (s, 1H),8.64 (s, 1H), 4.28 (s, 3H), 3.11 (s, 3H).

Example 6. Synthesis of4-methyl-2-(methylsulfonyl)-4,6-dihydro-5H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazin-5-one

Three necked flask charged with4-methyl-4,6-dihydro-5H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazin-5-one(30 g, 0.119 mol, 1.0 eq) in DCM (600 mL) m-CPBA (61.5 g, 3 eq) wasadded at 20° C. in three portions. The mixture was stirred at 30° C.overnight, LC-MS indicated 100% consumption of starting material, 20% ofE4-2 and 80% of E6-1 were formed. The mixture was cooled to r.t, anotherportion of m-CPBA (1.0 eq) was added. The reaction mixture was stirredat 30° C. for 2 hr, LC-MS indicated E4-2 (LCMS: m/z 269 (M+H)⁺.)<8%. Themixture was cooled to r.t. and filtered. The filtered cake wassuspension in MeOH (500 mL) and stirred at r.t. for 1 hr. Solid wascollected by filtration, washed with ethylacetate, dried in vacuum toafford 28 g of mixture of 5% of E4-2 and 95% of E6-1. The mixture (28 g)was suspended in DMSO (600 mL), heated to 120° C.-130° C. to form aclear solution. Then cooled to r.t., solid precipitated. The mixture wasfiltered and dried to provide 23 g of pure E6-1, LCMS: m/z 285 (M+H)⁺.1H NMR (400 MHz, DMSO) δ 12.87 (s, 1H), 8.69 (s, 1H), 4.32 (s, 3H), 3.56(s, 3H).

Example 7. Synthesis of Compounds E7-v and E7-viii

Nucleophilic aromatic substitution between compound E7-i and compoundE4-2, and/or compound E6-1, gives intermediate E7-ii. Reduction of thephenylsulfonyl group of compound E7-ii affords intermediate E7-iii.Using standard alkylation reaction of E7-iv and compound E7-iii in thepresence of base (e.g. K₂CO₃, K₃PO₄, t-BuOK, or Cs₂CO₃) gives compoundE7-vi, wherein X^(a) is a leaving group such as Cl, Br, I, OMs, OTs; Ar₁and Ar₂ are each independently optionally substituted 5-membered or6-membered monocyclic heteroaryl. Compound E7-vi can also be synthesizedfrom Mitsunobu reaction of compound E7-iii with E7-v usingCyanomethylene-tributylphosphorane (CMBP) in toluene.

Example 7A. Synthesis of4-methyl-2-((1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-3-yl)methyl)-4,6-dihydro-5H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazin-5-one

Step A.4-methyl-2-((phenylsulfonyl)(1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-3-yl)methyl)-4,6-dihydro-5H-thiazolo[5′,4′:4,5]pyrrolo[23-d]pyridazin-5-one

To a solution of3-((phenylsulfonyl)methyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-H-pyrazole(26 g, 78.86 mmol, 2.1 eq) in anhydrous THF (700 mL) was added LiHMDS(81.2 mL, 81.2 mmol, 1 M in THF, 2.31 eq) at −40° C. The reaction waswarmed up to room temperature and stirred for 1 hr. Then4-methyl-2-(methylsulfonyl)-4H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one(10 g, 35.17 mmol, 1 eq) was added to the mixture at room temperature.The reaction was stirred for another 0.5 hr at room temperature. Thereaction was poured into saturated ammonium chloride solution (500 mL)at 0° C. and extracted with ethyl acetate twice. The combined organiclayers were washed with water and brine, dried and concentrated to givea yellow residue. The residue was purified by silica gel chromatography(dichloromethane:methanol=100:1 to 30:1) to give4-methyl-2-((phenylsulfonyl)(1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-3-yl)methyl)-4H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one(13 g). LC-MS ESI M/Z=557 (M+H)⁺. 1H NMR (400 MHz, DMSO) δ 12.81 (s,1H), 8.65 (s, 1H), 8.03 (d, 1H), 7.84-7.78 (m, 3H), 7.66 (t, 2H), 6.94(s, 1H), 6.72 (d, 1H), 5.50 (d, 2H), 4.29 (s, 3H), 3.56 (dd, 2H), 0.91(dd, 2H), 0.02 (s, 9H).

Step B.4-methyl-2-((1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-3-yl)methyl)-4,6-dihydro-5H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazin-5-one

To a solution of4-methyl-2-((phenylsulfonyl)(1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-3-yl)methyl)-4H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one(8 g, 14.37 mmol, 1 eq) in THF (500 mL) and MeOH (500 mL) was added SmI₂(503 mL, 50.29 mmol, 0.1 M in THF, 3.5 eq) at room temperature. Thereaction was stirred for 0.5 hr and concentrated. The residue waspurified by silica gel chromatography (Petroleum ether:Ethylacetate:Methanol=75:25:0 to 20:20:1) to give4-methyl-2-((1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-3-yl)methyl)-4H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one(4.3 g). LC-MS ESI M/Z=417 (M+H)⁺. ¹H NMR (400 MHz, CDCl₃) δ 8.31 (s,1H), 7.60 (d, 1H), 7.28 (s, 1H), 6.39 (d, 1H), 5.49 (s, 2H), 4.58 (s,2H), 4.43 (s, 3H), 3.62 (t, 2H), 0.95 (t, 2H), 0.0 (s, 9H).

Example 7B. Synthesis of6-((1H-imidazol-2-yl)methyl)-2-((1H-pyrazol-3-yl)methyl)-4-methyl-4H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one

Step A. 1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazole-2-carbaldehyde

A sample of NaH was washed with hexane (2×10 mL) under N₂. The flask wascharged dry DMF (20 mL) and 1H-imidazole-2-carbaldehyde (500 mg, 5.2mmol) was added in small portions. After stirring at room temperaturefor 1.5 h, SEMCl (864 mg, 5.2 mmol) was added dropwise. The reactionmixture was stirred at r.t. for 30 min. The reaction mixture was pouredinto water, extracted with EtOAc. The organic layer was washed withbrine, dried over Na₂SO₄, concentrated under reduced pressure to affordcrude 1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazole-2-carbaldehyde(800 mg). LCMS: 227 (M+H)⁺.

Step B. (1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazol-2-yl)methanol

To a stirred mixture of1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazole-2-carbaldehyde (1.6 g,7 mmol) in THF (20 mL) was added NaBH₄ (1.34 g, 35 mmol) at 0° C. Thereaction mixture was stirred at r.t for 30 min. The reaction mixture waspoured into aq. NH₄Cl and extracted with EtOAc. The organic layer waswashed with brine, dried over Na₂SO₄, concentrated under reducedpressure to afford crude(1-((2-(trimethylsilyl)ethoxy)methyl)-H-imidazol-2-yl)methanol (1.3 g).

Step C.2-(chloromethyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazole

To a stirred mixture of(1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazol-2-yl)methanol (400 mg,1.75 mmol) in DCM (20 mL) were added NCS (466 mg, 3.5 mmol) and PPh₃(920 mg, 3.5 mmol) at r.t. The mixture was stirred at r.t for 2 h. Thereaction mixture was poured into water and extracted with DCM. Themixture was washed with water and the organic layer was concentratedunder reduced pressure. The residue was purified by Pre-TLC(PE:EtOAc=1:1) to afford2-(chloromethyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazole.LCMS: 247 (M+H)⁺. To a stirred mixture of4-methyl-2-((1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-3-yl)methyl)-4H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one(50 mg, 0.12 mmol) in dry DMF (5 mL) was added K₂CO₃ (66 mg, 0.48 mmol)at 60° C. under N₂. After 20 min, compound E7-2 (60 mg, 0.24 mmol), indry DMF (2 mL) was added at 60° C. under N₂. The mixture was stirred at60° C. for 1.5 h under N₂. The reaction mixture was cooled to r.t andadjusted at pH=5-6 with 0.5N aq. HCl. Then the mixture was extractedwith EtOAc, washed with water and brine. The organic layer was driedover Na₂SO₄, concentrated under reduced pressure and purified byPrep-TLC (PE:EtOAc=1:1.5) to afford4-methyl-6-((1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazol-2-yl)methyl)-2-((1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-3-yl)methyl)-4H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one(25 mg). LCMS: 627 (M+H)⁺. A mixture of4-methyl-6-((1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazol-2-yl)methyl)-2-((1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-3-yl)methyl)-4H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one(25 mg, 0.04 mmol) in DCM/TFA (2 mL/2 mL) was stirred at r.t for 1 hour.The reaction mixture was concentrated. The residue was purified byprep-HPLC to afford desired product (1.3 mg). LCMS: 367 (M+H)⁺. ¹H NMR(400 MHz, DMSO-d₆) δ 8.51 (s, 1H), 7.66 (d, 1H), 6.9 (s, 2H), 6.27 (d,1H), 5.34 (s, 2H), 4.51 (s, 2H), 4.27 (s, 3H).

The following compounds were synthesized according to Scheme E7 and theprocedure of Example 7A-7B using the appropriate starting material.Standard protection and deprotection methods are used when necessary.

Cpd No. Structure and chemical name Characterization E7-6

LCMS: 384 (M + H)⁺. ¹H NMR (400 MHz, DMSO) δ 12.78 (s, 1H), 9.03 (d,1H), 8.52 (s, 1H), 7.71 (s, 1H), 7.42 (d, 1H), 6.27 (d, 1H), 5.48 (s,2H), 4.48 (s, 2H), 4.27 (s, 3H). E7-7

LCMS: 385 (M + H)+. 1HNMR (400 MHz, DMSO) δ 12.79 (s, 1H), 8.84 (s, 1H),8.64 (s, 1H), 7.72 (s, 1H), 6.27 (d, 1H), 5.84 (s, 2H), 4.51 (s, 2H),4.28 (s, 3H) E7-8

LCMS: m/z 399 (M + H)+. 1H NMR (400 MHz, DMSO-d6) δ 12.78 (s, 1H), 8.52(s, 1H), 7.70 (s, 1H), 6.91 (s, 1H), 6.87 (br s, 2H), 6.27 (d, 1H), 5.26(s, 2H), 4.49 (s, 2H), 4.27 (s, 3H). E7-9

LCMS: m/z 367 (M + H)+. 1H NMR (400 MHz, DMSO-d6) δ 8.48 (s, 1H), 7.68(s, 1H), 7.52 (s, 1H), 6.91 (s, 1H), 6.27 (d, 1H), 5.25 (s, 2H), 4.50(s, 2H), 4.28 (s, 3H). E7-10

LC-MS: m/z 367 (M + H)⁺. 1H NMR (400 MHz, DMSO) δ 12.77 (brs, 2H), 8.49(s, 1H), 7.72- 7.35 (m, 3H), 6.25 (s, 1H), 5.20 (s, 2H), 4.48 (s, 2H),4.27 (s, 3H). E7-11

LC-MS: m/z 384 (M + 1)⁺. 1H NMR (400 MHz, DMSO) δ 12.77 (s, 1H), 8.59(s, 1H), 8.46 (d, 1H), 7.71 (s, 1H), 7.42 (d, 1H), 6.26 (d, 1H), 5.66(s, 2H), 4.48 (s, 2H), 4.28 (s, 3H). E7-12

LCMS: m/z 414 (M + H)⁺. 1H NMR (400 MHz, DMSO) δ 12.78 (s, 1H), 8.48 (s,1H), 7.70 (s, 1H), 7.28 (s, 1H), 6.26 (d, 1H), 6.15 (t, 1H), 5.39 (s,2H), 4.66 (d, 2H), 4.49 (s, 2H), 4.25 (s, 3H). E7-13

LCMS: m/z 427 (M + H)+. 1H NMR (400 MHz, DMSO) δ 12.78 (s, 1H), 8.51 (s,1H), 8.11 (s, 1H), 7.78 (s, 1H), 7.70-7.60 (m, 2H), 6.27 (d, 1H), 5.50(s, 2H), 4.48 (s, 2H), 4.26 (s, 3H). E7-14

LCMS: m/z 368 (M + H)⁺. 1H NMR (400 MHz, DMS0-d6) δ 14.89 (s, 1H), 12.78(s, 1H), 8.51 (s, 1H), 7.95-7.54 (m, 2H), 6.26 (d, 1H), 5.42 (s, 2H),4.49 (s, 2H), 4.27 (s, 3H). E7-15

LCMS: (ESI) m/z 453 (M + H)⁺. ¹H NMR (400 MHz, DMSO-d₆) δ 12.84 (s, 1H),10.41 (s, 1H), 8.57 (s, 1H), 8.06 (s, 1H), 7.71 (s, 2H), 6.34 (s, 1H),5.52 (s, 2H), 4.58 (s, 2H), 4.33 (s, 3H), 2.08 (s, 3H). E7-16

LC-MS: 384 [M + H]+. 1H NMR (400 MHz, DMSO) δ 12.78 (s, 1H), 8.58 (s,1H), 7.75 (d, 1H), 7.70 (s, 1H), 7.64 (d, 1H), 6.27 (s, 1H), 5.65 (s,2H), 4.48 (s, 2H), 4.27 (s, 3H). E7-17

LCMS: m/z 462, 464 (M, M + 2H)⁺. ¹H NMR (400 MHz, DMSO-d₆) δ 12.77 (s,1H), 9.04 (s, 1H), 8.49 (s, 1H), 7.71 (s, 1H), 6.26 (d, 1H), 5.41 (s,2H), 4.48 (s, 2H), 4.25 (s, 3H). E7-18

LCMS: m/z 399 (M + H)⁺. ¹H NMR (400 MHz, DMSO-d₆) δ 12.78(s, 1H), 8.51(s, 1H), 7.99 (s, 1H), 7.71 (s, 1H), 6.27 (d, 1H), 5.83 (s, 2H), 5.27(s, 2H), 4.49 (s, 2H), 4.27 (s, 3H) E7-19

LCMS: m/z 409 (M + H)⁺. ¹H NMR (400 MHz, DMSO-d₆) δ 12.77 (s, 1H), 9.36(s, 1H), 8.56 (s, 1H), 7.71 (s, 1H), 6.26 (d, 1H), 5.61 (s, 2H), 4.49(s, 2H), 4.25 (s, 3H). E7-20

LCMS: m/z 427 (M + H)⁺. ¹H NMR (400 MHz, DMSO-d₆) δ 12.78 (s, 1H), 8.97(s, 1H), 8.51 (s, 1H), 7.70 (s, 1H), 7.75 (d, 2H), 6.26 (d, 1H), 5.67(s, 2H), 4.49 (s, 2H), 4.25 (s, 3H). E7-21

LCMS: m/z 398 (M + H)⁺. ¹H NMR (400 MHz, DMSO-d₆) δ 12.77 (s, 1H), 8.74(s, 1H), 8.47 (s, 1H), 7.70 (s, 1H), 6.26 (d, 1H), 5.39 (s, 2H), 4.49(s, 2H), 4.26 (s, 3H), 2.49 (s, 3H). E7-22

LCMS: 421 (M + H)⁺. ¹H NMR (400 MHz, DMSO) δ 12.78 (s, 1H), 8.53 (s,1H), 7.68 (s, 1H), 7.39 (dd, 1H), 6.47 (d, 1H), 6.27 (d, 1H), 6.17 (d,1H), 5.27 (s, 2H), 4.47 (m, 2H), 4.26 (s, 3H), 2.94 (s, 6H). E7-23

LCMS: m/z 452 (M + H)+. ¹H NMR (400 MHz, DMSO) δ 12.85 (s, 1H), 9.24 (s,1H), 8.50 (s, 1H), 7.67 (s, 1H), 6.27 (d, 1H), 5.57 (s, 2H), 4.50 (s,2H), 4.25 (s, 3H). E7-24

LCMS: m/z 418 (M + H)+. ¹H NMR (400 MHz, DMSO) δ 12.79 (s, 1H), 8.94 (s,1H), 8.49 (s, 1H), 7.70 (s, 1H), 6.26 (d, 1H), 5.42 (s, 2H), 4.49 (s,2H), 4.26 (s, 3H). E7-25

LCMS (ESI): m/z 414 (M + H)⁺. ¹H NMR DMS0-d6 400 MHz δ 8.45 (s, 1H),8.41 (s, 1H), 8.29 (s, 1H), 7.66 (s, 1H), 6.26 (d, 1H), 5.30 (s, 2H),4.49 (s, 2H), 4.26 (s, 3H), 3.93 (s, 3H). E7-26

LCMS (ESI): m/z 378 (M + H). ¹H NMR (DMSO-d6 400 MHz) δ 12.79 (s, 1H),8.57 (d, 1H), 8.54 (s, 1H), 8.48 (dd, 1H), 7.71 (ddd, 2H), 7.35 (ddd,1H), 6.26 (d, 1H), 5.38 (s, 2H), 4.49 (s, 2H), 4.26 (s, 3H). E7-27

LCMS: m/z 423 (M + H)⁺. ¹H NMR (400 MHz, DMSO-d₆) δ 12.78 (s, 1H), 8.51(s, 1H), 7.71 (s, 1H), 6.91 (d, 1H), 6.26 (d, 1H), 6.17 (d, 1H), 5.68(s, 2H), 5.16 (s, 2H), 4.49 (s, 2H), 4.27 (s, 3H), 3.72 (s, 3H). E7-28

LCMS: m/z 411 (M + H)⁺. ¹H NMR (400 MHz, DMSO-d₆) δ 8.56 (s, 1H), 7.67(s, 1H), 6.3-6.2 (m, 2H), 6.18 (d, 1H), 5.27 (s, 2H), 4.51 (s, 2H), 4.27(s, 3H). E7-29

LCMS: m/z 426 (M + H)⁺. ¹H NMR (400 MHz, DMSO) δ 8.56 (s, 1H), 7.73 (d,1H), 7.67 (s, 1H), 7.08 (d, 1H), 6.26 (d, 1H), 5.39 (s, 2H), 4.50 (s,2H), 4.26 (s, 3H), 2.30 (s, 3H). E7-30

LCMS: m/z 385 (M + H)⁺. ¹H NMR (400 MHz, DMSO) δ 12.83 (s, 1H), 12.68(s, 1H), 8.46 (s, 1H), 7.64 (s, 2H), 6.26 (s, 1H), 5.33 (s, 2H), 4.48(s, 2H), 4.24 (s, 3H). E7-31

LCMS: m/z 394 (M + H)⁺. ¹H NMR (400 MHz, DMSO) δ 12.79 (s, 1H), 11.74(s, 1H), 8.55 (s, 1H), 7.72 (s, 1H), 7.30 (d, 1H), 6.83 (d, 1H), 6.27(d, 1H), 6.09 (t, 1H), 5.10 (s, 2H), 4.51 (s, 2H), 4.27 (s, 3H). E7-32

LCMS: m/z 408 (M + H)⁺. ¹H NMR (400 MHz, DMSO) δ 12.78 (s, 1H), 8.56 (s,1H), 8.08 (dd, 1H), 7.70 (s, 1H), 7.16 (d, 1H), 6.90 (dd, 1H), 6.27 (d,1H), 5.29 (s, 2H), 4.51 (s, 2H), 4.27 (s, 3H), 3.93 (s, 3H). E7-33

LCMS: m/z 385 (M + H)⁺. ¹H NMR (400 MHz, DMSO-d₆) δ 12.88 (s, 1H), 12.61(s, 1H), 8.61 (d, 1H), 7.78 (s, 1H), 6.33 (d, 1H), 5.89 (d, 1H), 5.36(s, 2H), 4.54 (d, 2H), 4.34 (s, 3H). E7-34

LCMS: m/z 407 (M + H)⁺. ¹H NMR (400 MHz, DMSO-d₆) δ 12.84 (s, 1H), 8.58(s, 1H), 7.79 (s, 1H), 7.18 (d, 1H), 6.33 (s, 1H), 6.15 (d, 1H), 5.75(s, 2H), 5.25 (s, 2H), 4.55 (s, 2H), 4.33 (s, 3H), 2.05 (s, 3H). E7-35

LC-MS: m/z 408 (M + H)⁺. ¹H NMR (400 MHz, DMSO) δ 12.87 (s, 1H), 8.60(s, 1H), 8.27 (d, 1H), 7.84-7.72 (m, 2H), 6.86 (d, 1H), 6.34 (d, 1H),5.37 (s, 2H), 4.55 (s, 2H), 4.34 (s, 3H), 3.89 (s, 3H). E7-36

LC-MS: m/z 394 (M + H)⁺. ¹H NMR (400 MHz, DMSO) δ 12.77 (s, 1H), 11.53(s, 1H), 8.52 (s, 1H), 7.69 (s, 1H), 7.47 (dd, 1H), 7.40 (d, 1H), 6.27(dd, 2H), 5.07 (s, 2H), 4.49 (s, 2H), 4.26 (s, 3H). E7-37

LC-MS: m/z 408 (M + H)⁺. ¹H NMR (400 MHz, DMSO) δ 12.86 (s, 1H), 8.65(d, 1H), 8.17 (d, 1H), 7.80 (s, 1H), 6.94-6.89 (m, 1H), 6.66 (s, 1H),6.34 (d, 1H), 5.41 (s, 2H), 4.56 (s, 2H), 4.34 (s, 3H), 3.89 (s, 3H).E7-38

LC-MS: m/z 394 (M + H)⁺. ¹H NMR (400 MHz, DMSO) δ 8.57 (s, 1H), 7.68 (s,1H), 7.31 (d, 1H), 7.23 (t, 1H), 6.27 (d, 1H), 6.07 (dd, 1H), 5.97 (s,1H), 5.17 (s, 2H), 4.51 (s, 2H), 4.27 (s, 3H). E7-39

LCMS: m/z 382 (M + H)⁺. ¹HNMR (400 MHz, DMSO) δ 12.80 (s, 1H), 8.46 (s,1H), 8.39 (s, 1H), 7.66 (s, 1H), 6.34 (s, 1H), 6.26 (d, 1H), 5.18 (s,2H), 5.06 (s, 2H), 4.49 (s, 2H), 4.27 (s, 3H). E7-40

LCMS: m/z 409 (M + H)⁺. ¹HNMR (400 MHz, DMSO) δ 12.83 (s, 1H), 8.74 (s,1H), 8.55-8.65 (m, 2H), 7.79 (s, 1H), 7.56 (t, 1H), 6.77 (d, 1H), 6.40(d, 1H), 6.32 (d, 1H), 5.32 (s, 2H), 4.52 (s, 2H), 4.31 (s, 3H) E7-41

LCMS: ESI m/z 384 (M + H)⁺. ¹H NMR (400 MHz, DMSO-d₆) δ 9.01 (d, 1H),8.54 (s, 1H), 7.67 (d, 1H), 7.21 (d, 1H), 6.27 (d, 1H), 5.50 (s, 2H),4.51 (s, 2H), 4.26 (s, 3H). E7-42

LCMS: ESI m/z 382 (M + H)⁺. ¹H NMR (400 MHz, DMSO-d₆) δ 12.78 (s, 1H),8.55 (s, 1H), 7.71 (s, 1H), 7.62 (s, 1H), 6.26 (d, 1H), 5.49 (s, 2H),4.48 (s, 2H), 4.26 (s, 3H), 4.10 (s, 3H). E7-43

LCMS: ESI m/z 382 (M + H)⁺. ¹H NMR (400 MHz, DMSO-d₆) δ 12.78 (s, 1H),8.51 (s, 1H), 7.96 (s, 1H), 7.71 (s, 1H), 6.27 (d, 1H), 5.38 (s, 2H),4.49 (s, 2H), 4.27 (s, 3H), 3.99 (s, 3H). E7-44

LCMS: ESI m/z 378 (M + H)⁺. ¹H NMR (400 MHz, DMSO-d₆) δ 12.94 (s, 1H),8.64 (s, 1H), 8.57 (d, 1H), 7.82 (m, 2H), 7.36 (m, 1H), 7.23 (d, 1H),6.36 (d, 1H), 5.55 (s, 2H), 4.58 (s, 2H), 4.36 (s, 3H). E7-45

LCMS: m/z 401 (M + H)⁺. ¹H NMR (400 MHz, DMSO-d6) δ 12.96 (s, 1H), 12.77(s, 1H), 8.48 (s, 1H), 7.91 (s, 1H), 7.71 (s, 1H), 6.26 (d, 1H), 5.33(s, 2H), 4.49 (s, 2H), 4.27 (s, 3H). E7-46

LCMS: m/z 410 (M + H)⁺. ¹H NMR (400 MHz, DMSO-d6) δ 13.34 (s, 1H), 12.81(s, 1H), 8.54 (s, 1H), 7.70 (s, 2H), 7.26 (m, 1H), 6.64 (s, 1H), 6.27(s, 1H), 5.35 (s, 2H), 4.50 (s, 2H), 4.28 (s, 3H) E7-47

LC-MS m/z 411.0 (M + H)⁺. ¹H NMR (400 MHz, DMSO) δ 12.79 (s, 1H), 8.46(s, 1H), 7.73 (s, 1H), 7.29 (dd, 1H), 6.34 (dd, 1H), 6.27 (d, 1H), 5.74(s, 2H), 5.30 (s, 2H), 4.46 (s, 2H), 4.26 (s, 3H) E7-48

LC-MS m/z 382.0 (M + H)⁺. ¹H NMR (400 MHz, DMSO) δ 12.79 (s, 1H), 8.51(s, 1H), 7.68 (s, 1H), 7.68 (s, 1H), 6.26 (d, 1H), 5.38 (s, 2H), 4.50(s, 2H), 4.26 (s, 3H), 4.08 (s, 3H) E7-49

LC-MS m/z 425 (M + H)⁺. ¹H NMR (400 MHz, DMSO) δ 12.77 (s, 1H), 8.47 (s,1H), 7.71 (s, 1H), 7.31 (t, 1H), 6.33-6.26 (m, 3H), 5.34 (s, 2H), 4.49(s, 2H), 4.26 (s, 3H), 2.48 (s, 3H) E7-50

LCMS: (ESI) m/z 411 (M + H)⁺. ¹H NMR (400 MHz, DMSO-d₆) δ 8.59 (s, 1H),7.73 (s, 1H), 7.29 (dd, 1H), 6.47-6.06 (m, 4H), 5.26 (s, 2H), 4.56 (s,2H), 4.32 (s, 3H) E7-51

LCMS: (ESI) m/z 378 (M + H)⁺. ¹H NMR (400 MHz, DMSO-d₆) δ 12.84 (s, 1H),8.64 (s, 1H), 8.57 (d, 1H), 8.55 (d, 1H), 7.77 (s, 1H), 7.28 (d, 2H),6.33 (d, 1H), 5.45 (s, 2H), 4.56 (s, 2H), 4.32 (s, 3H) E7-52

LCMS: (ESI) m/z 384 (M + H)⁺. ¹H NMR (400 MHz, DMSO-d₆) δ 12.85 (s, 1H),9.01 (s, 1H), 8.54 (d, 1H), 7.95 (d, 1H), 7.74 (s, 1H), 6.24 (s, 1H),5.55 (s, 2H), 4.46 (s, 2H), 4.30 (s, 3H) E7-53

LCMS: (ESI) m/z 392 (M + H)⁺. ¹H NMR (400 MHz, DMSO-d₆) δ 12.78 (s, 1H),8.55 (s, 1H), 7.74 (s, 1H), 7.61 (dd, 1H), 7.14 (d, 1H), 6.81 (d, 1H),6.26 (s, 1H), 5.39 (s, 2H), 4.50 (s, 2H), 4.26 (s, 3H), 2.43 (s, 3H)E7-54

LCMS: (ESI) m/z 384 (M + H)⁺. ¹H NMR (400 MHz, DMSO-d₆) δ 12.78 (s, 1H),8.91 (s, 1H), 8.56 (s, 1H), 8.53 (s, 1H), 7.71 (s, 1H), 6.26 (s, 1H),5.48 (s, 2H), 4.49 (s, 2H), 4.31 (s, 3H) E7-55

LCMS: (ESI) m/z 368 (M + H)⁺. ¹H NMR (400 MHz, DMSO-d₆) δ 13.85 (s, 1H),12.78 (s, 1H), 8.51 (s, 1H), 8.49-8.21 (m, 1H), 7.71 (s, 1H), 6.27 (d,1H), 5.41 (s, 2H), 4.49 (s, 2H), 4.26 (s, 3H) E7-56

LCMS: (ESI) m/z 385 (M + H)⁺. ¹H NMR (400 MHz, DMSO-d₆) δ 12.79 (s, 1H),9.57 (s, 1H), 8.61 (s, 1H), 7.71 (s, 1H), 6.26 (d, 1H), 5.80 (s, 2H),4.49 (s, 2H), 4.27 (s, 3H) E7-57

LCMS: (ESI) m/z 476 (M + 2H)⁺. ¹H NMR (400 MHz, DMSO-d₆) δ 12.78 (s,1H), 8.57 (s, 1H), 7.81 (dd, 1H), 7.71 (s, 1H), 7.30 (dd, 1H), 6.26 (d,1H), 5.43 (s, 2H), 4.49 (s, 2H), 4.26 (s, 3H) E7-58

LCMS: (ESI) m/z 401 (M + H)⁺. ¹H NMR (400 MHz, DMSO-d₆) δ 13.01 (s, 1H),12.95 (s, 1H), 8.53 (s, 1H), 7.68 (dd, 1H), 6.26 (d, 1H), 6.17 (s, 1H),5.32 (s, 2H), 4.50 (s, 2H), 4.26 (s, 3H) E7-59

LCMS: (ESI) m/z 399 (M + H)⁺. ¹H NMR (400 MHz, DMSO-d₆) δ 12.77 (s, 1H),8.47 (s, 1H), 7.75 (d, 1H), 7.71 (s, 1H),6.26 (s, 1H), 5.29 (s, 2H),4.47 (s, 2H), 4.26 (s, 3H), 3.70 (s, 3H) E7-60

LCMS: (ESI) m/z 415 (M + H)⁺. ¹H NMR (400 MHz, DMSO-d₆) δ 12.82 (s, 1H),8.52 (s, 1H), 7.92 (s, 1H), 7.76 (s, 1H), 6.31 (s, 1H), 5.34 (s, 2H),4.53 (s, 2H), 4.31 (s, 3H), 3.79 (s, 3H) E7-61

LCMS: (ESI) m/z 399 (M + H)⁺. ¹H NMR (400 MHz, DMSO-d₆) δ 12.79 (s, 1H),8.49 (s, 1H), 7.71 (s, 1H), 6.91 (s, 2H), 6.27 (s, 1H), 6.18 (s, 1H),5.12 (s, 2H), 4.49 (s, 2H), 4.27 (s, 3H). E7-62

LCMS: (ESI) m/z 408 (M + H)⁺. ¹H NMR (400 MHz, DMSO-d₆) δ 12.79 (s, 1H),8.56 (s, 1H), 7.71 (s, 1H), 7.63 (dd, 1H), 6.68 (d, 1H), 6.60 (d, 1H),6.27 (d, 1H), 5.36 (s, 2H), 4.51 (s, 2H), 4.26 (s, 3H), 3.76 (s, 3H).E7-63

LC-MS: m/z 406 (M + H)⁺. ¹H NMR (400 MHz, DMSO-d6) δ: 12.79 (s, 1H),8.53 (s, 1H), 8.44 (s, 1H), 7.72 (s, 1H), 6.27 (s, 1H), 5.40 (s, 2H),4.48 (s, 2H), 4.26 (s, 3H), 3.83 (s, 3H). E7-64

LC-MS: m/z 424 (M + H)⁺. ¹H NMR (400 MHz, DMSO-d6) δ: 12.79 (s, 1H),8.46 (s, 1H), 8.10 (s, 1H), 7.72 (s, 1H), 7.57 (s, 1H), 7.02 (s, 1H),6.27 (s, 1H), 5.53 (s, 2H), 4.48 (s, 2H), 4.26 (s, 3H), 3.69 (s, 3H).E7-65

LC-MS: m/z 396 (M + H)⁺. ¹H NMR (400 MHz, DMSO-d6) δ: 8.51 (s, 1H), 7.67(s, 1H), 6.95 (s, 1H), 6.27 (d, 1H), 5.17 (s, 2H), 4.50 (s, 2H), 4.27(s, 3H), 3.61 (s, 3H). E7-66

LC-MS: m/z 392 (M + H)⁺. ¹H NMR (400 MHz, DMSO-d6) δ: 13.62 (s, 1H),12.79 (s, 1H), 8.54 (m, 2H), 7.71 (s, 1H), 6.27 (d, 1H), 5.45 (s, 2H),4.49 (s, 2H), 4.26 (s, 3H). E7-67

LC-MS: m/z 410 (M + H)⁺. ¹H NMR (400 MHz, DMSO-d6) δ: 12.75 (s, 2H),8.48 (s, 1H), 8.17 (s, 1H), 7.68 (s, 1H), 7.56 (s, 1H), 7.01 (s, 1H),6.27 (d, 1H), 5.57 (s, 2H), 4.50 (s, 2H), 4.26 (s, 3H). E7-68

LC-MS: m/z 449 (M + H)⁺. ¹H NMR (400 MHz, DMSO-d6) δ: 12.85 (s, 1H),8.54 (s, 1H), 8.34 (d, 1H), 7.77 (s, 1H), 6.32 (s, 1H), 5.44 (d, 2H),4.54 (s, 2H), 4.32 (s, 3H), 3.83 (s, 3H). E7-69

LC-MS: m/z 411 (M + H)⁺. ¹H NMR (400 MHz, DMSO-d6) δ: 12.77 (s, 1H),8.43 (s, 1H), 7.71 (s, 1H), 7.41 (s, 1H), 6.26 (d, 1H), 5.20 (s, 2H),4.48 (s, 2H), 4.26 (s, 3H), 3.65 (s, 3H), 3.61 (s, 3H). E7-70

LC-MS: m/z 399 (M + H)⁺. ¹H NMR (400 MHz, DMSO-d6) δ: 12.90- 12.30 (m,2H), 8.49 (s, 1H), 7.81 (s, 1H), 7.34 (s, 1H), 5.30 (s, 2H), 4.50 (s,2H), 4.26 (s, 3H), 1.96 (s, 3H). E7-71

LC-MS: m/z 402 (M + H)⁺. ¹H NMR (400 MHz, DMSO-d6) δ: 12.80 (s, 1H),9.04 (d, 1H), 8.54 (s, 1H), 7.88 (s, 1H), 7.49-7.39 (m, 1H), 5.48 (s,2H), 4.50 (s, 2H), 4.26 (s, 3H). E7-72

LC-MS: m/z 395 (M + H)⁺. ¹H NMR (400 MHz, DMSO-d6) δ: 12.86 (s, 1H),8.51 (s, 1H), 7.72 (s, 1H), 7.41 (s, 1H), 6.32 (s, 1H), 5.29 (s, 2H),4.55 (s, 2H), 4.32 (s, 3H), 3.74 (s, 3H), 1.99 (s, 3H). E7-73

LC-MS: m/z 400 (M + H)⁺. ¹H NMR (400 MHz, DMSO-d6) δ: 8.53 (s, 1H), 7.80(s, 1H), 6.96 (s, 1H), 5.23 (s, 2 H), 4.50 (s, 2 H), 4.27 (s, 3H). E7-74

LC-MS: m/z 411 (M + H)⁺. ¹H NMR (400 MHz, DMSO-d6) δ: 12.82 (s, 1H),8.48 (s, 1H), 7.67 (s, 1H), 7.50 (s, 1H), 6.27 (d, 1H), 5.28 (s, 2H),4.50 (s, 2H), 4.38 (s, 2H), 4.27 (s, 3H), 3.71 (s, 3H). E7-75

LC-MS: m/z 411 (M + H)⁺. ¹H NMR (400 MHz, DMSO-d6) δ: 12.80 (s, 1H),8.49 (s, 1H), 8.38 (s, 1H), 8.09 (s, 1H), 7.80 (s, 1H), 7.72 (s, 1H),6.27 (d, 1H), 5.56 (s, 2H), 4.49 (s, 2H), 4.26 (s, 3H). E7-76

LC-MS: m/z 403 (M + H)⁺. ¹H NMR (400 MHz, DMSO-d6) δ: 12.82 (s, 1H),8.94 (d, 1H), 8.57 (s, 1H), 8.26 (dd, 1H), 7.69 (s, 1H), 7.42 (d, 1H),6.27 (d, 1H), 5.55 (s, 2H), 4.51 (s, 2H), 4.25 (s, 3H). E7-77

LC-MS: m/z 421 (M + H)⁺. ¹H NMR (400 MHz, DMSO-d6) δ: 12.82 (s, 1H),8.92 (d, 1H), 8.56 (s, 1H), 8.14 (dd, 2H), 7.69 (s, 1H), 7.57 (s, 1H),7.23 (d, 1H), 6.27 (d, 1H), 5.50 (s, 2H), 4.51 (s, 2H), 4.26 (s, 3H).E7-78

LC-MS: m/z 421 (M + H)⁺. ¹H NMR (400 MHz, DMSO-d6) δ: 12.79 (s, 1H),8.59 (s, 1H), 7.97-7.82 (m, 3H), 7.75-7.60 (m, 2H), 7.26 (dd, 1H), 6.27(d, 1H), 5.53 (s, 2H), 4.51 (s, 2H), 4.26 (s, 3H). E7-79

LC-MS: m/z 403 (M + H)⁺. ¹H NMR (400 MHz, DMSO-d6) δ: 8.64 (s, 1H),8.12-7.98 (m, 2H), 7.74 (s, 1H), 7.60 (d, 1H), 6.33 (d, 1H), 5.57 (s,2H), 4.57 (s, 2H), 4.32 (s, 3H). E7-80

LC-MS: m/z 399 (M + H)⁺. ¹H NMR (400 MHz, DMSO-d6) δ: 12.79 (s, 1H),8.55 (s, 1H), 7.68 (s, 1H), 6.89 (s, 1H), 6.27 (s, 1H), 5.58 (s, 2H),5.36 (s, 2H), 4.50 (s, 2H), 4.27 (s, 3H). E7-81

LC-MS: m/z 429 (M + H)⁺. ¹H NMR (400 MHz, DMSO-d6) δ: 12.84 (s, 1H),8.48 (s, 1H), 7.88 (d, 1H), 7.29 (t, 1H), 6.34 (dd, 1H), 5.76 (s, 2H),5.31 (s, 2H), 4.51 (s, 2H), 4.23 (s, 3H). E7-82

LC-MS: m/z 383 (M + H)⁺. ¹H NMR (400 MHz, DMSO-d6) δ: 12.85 (s, 1H),12.10 (s, 1H), 8.57 (s, 1H), 8.32 (s, 1H), 7.77 (s, 1H), 7.21 (s, 1H),6.33 (d, 1H), 5.32 (s, 2H), 4.55 (s, 2H), 4.34 (s, 3H). E7-83

LC-MS: m/z 415 (M + H)⁺. ¹H NMR (400 MHz, DMSO-d6) δ: 12.76 (s, 1H),12.13 (s, 1H), 8.45 (s, 1H), 7.87 (s, 1H), 7.39 (s, 1H), 5.25 (s, 2H),4.49 (s, 2H), 4.26 (s, 3H), 3.63 (s, 3H). E7-84

LC-MS: m/z 416 (M + H)⁺. ¹H NMR (400 MHz, DMSO-d6) δ: 12.78 (s, 1H),8.75 (s, 1H), 8.49 (s, 1H), 7.85 (s, 1H), 5.39 (s, 2H), 4.50 (s, 2H),4.26 (s, 3H), 2.51 (s, 3H). E7-85

LC-MS: m/z 409 (M + H)⁺. ¹H NMR (400 MHz, DMSO-d6) δ: 12.80 (s, 1H),8.82 (s, 1H), 8.63 (s, 1H), 7.69 (s, 1H), 6.27 (d, 1H), 5.69 (s, 2H),4.51 (s, 2H), 4.27 (s, 3H). E7-86

LC-MS: m/z 427 (M + H)⁺. ¹H NMR (400 MHz, DMSO-d6) δ: 8.63 (s, 1H), 8.19(s, 1H), 7.76-7.63 (m, 2H), 7.57 (s, 1H), 6.27 (s, 1H), 5.67 (s, 2H),4.51 (s, 2H), 4.28 (s, 3H). E7-87

LC-MS: m/z 436 (M + H)⁺. ¹H NMR (400 MHz, DMSO-d6) δ: 12.83 (s, 1H),8.94 (s, 1H), 8.51 (s, 1H), 7.83 (s, 1H), 5.43 (s, 2H), 4.51 (s, 2H),4.26 (s, 3H). E7-88

LC-MS: m/z 383 (M + H)⁺. ¹H NMR (400 MHz, DMSO-d6) δ: 12.79 (s, 1H),8.50 (s, 1H), 7.70 (s, 1H), 6.65 (s, 1H), 6.57 (s, 2H), 6.23 (d, 1H),5.21 (s, 2H), 4.49 (s, 2H), 4.27 (s, 3H). E7-89

LC-MS: m/z 465 (M + H). ¹H NMR (400 MHz, DMSO-d6) δ: 12.78 (s, 1H), 8.48(s, 1H), 7.99 (s, 1H), 7.72 (s, 1H), 6.27 (d, 1H), 5.31 (s, 2H), 4.49(s, 2H), 4.27 (s, 3H), 3.76 (s, 3H). E7-90

LC-MS: m/z 411 (M + H)⁺. ¹H NMR (400 MHz, DMSO-d6) δ: 12.78 (s, 1H),8.56 (s, 1H), 8.17 (s, 1H), 7.84 (s, 1H), 7.72 (s, 1H), 7.34 (s, 1H),6.27 (s, 1H), 5.48 (s, 2H), 4.49 (s, 2H), 4.27 (s, 3H). E7-91

LC-MS: m/z 402 (M + H)⁺. ¹H NMR (400 MHz, DMSO-d6) δ: 12.78 (s, 1H),8.57 (d, 1H), 8.50 (s, 1H), 7.71 (s, 1H), 6.26 (d, 1H), 5.38 (d, 2H),4.49 (s, 2H), 4.26 (s, 3H). E7-92

LC-MS: m/z 410 (M + H)⁺. ¹H NMR (400 MHz, DMSO-d6) δ: 12.83 (s, 1H),8.49 (s, 1H), 7.67 (s, 2H), 7.38 (s, 1H), 7.06 (s, 1H), 6.26 (d, 1H),5.30 (s, 2H), 4.49 (s, 2H), 4.27 (s, 3H). E7-93

LC-MS: m/z 402 (M + H)⁺. ¹H NMR (400 MHz, DMSO-d6) δ: 12.79 (s, 1H),8.52 (s, 1H), 8.17 (s, 1H), 7.71 (s, 1H), 6.26 (d, 1H), 5.23 (s, 2H),4.49 (s, 2H), 4.27 (s, 3H). E7-94

LC-MS: m/z 418 (M + H)⁺. ¹H NMR (400 MHz, DMSO-d6) δ: 12.82 (s, 1H),8.58 (s, 1H), 7.75 (s, 1H), 7.49 (s, 1H), 6.31 (d, 1H), 5.42 (s, 2H),4.54 (s, 2H), 4.31 (s, 3H). E7-95

LC-MS: m/z 382 (M + H)⁺. ¹H NMR (400 MHz, DMSO-d6) δ: 8.48 (s, 1H), 7.67(s, 1H), 7.37 (d, 1H), 6.34 (d, 2H), 6.27 (d, 1H), 6.00 (d, 1H), 5.24(s, 2H), 4.50 (s, 2H), 4.27 (s, 3H). E7-96

LC-MS: m/z 425 (M + H)⁺. ¹H NMR (400 MHz, DMSO-d6) δ: 12.79 (s, 1H),8.46 (s, 1H), 7.72 (s, 1H), 7.59 (s, 1H), 6.27 (s, 1H), 5.28 (s, 2H),4.48 (s, 2H), 4.29-4.25 (m, 5H), 3.73 (s, 3H), 3.11 (s, 3H). E7-97

LC-MS: m/z 402 (M + H)⁺. ¹H NMR (400 MHz, DMSO-d6) δ: 12.78 (s, 1H),8.52 (s, 1H), 8.39 (s, 1H), 7.68 (s, 1H), 6.27 (d, 1H), 5.24 (s, 2H),4.50 (s, 2H), 4.26 (s, 3H). E7-98

LC-MS: m/z 469 (M + H)⁺. ¹H NMR (400 MHz, DMSO-d6) δ: 13.37- 12.75 (m,2H), 8.65-8.50 (m, 1H), 8.15-7.58 (m, 2H), 5.49-5.36 (m, 2H), 4.67-4.50(m, 2H), 4.32 (s, 3H). E7-99

LC-MS: m/z 383 (M + H)⁺. ¹H NMR (400 MHz, DMSO-d6) δ: 12.84 (s, 1H),8.53 (s, 1H), 7.77 (s, 1H), 7.25 (s, 1H), 6.58 (s, 2H), 6.32 (d, 1H),5.10 (s, 2H), 4.54 (s, 2H), 4.32 (s, 3H). E7-100

LC-MS: m/z 410 (M + H)⁺. ¹H NMR (400 MHz, DMSO-d6) δ: 12.89 (s, 1H),8.58 (s, 1H), 8.45 (s, 1H), 7.77- 7.64 (m, 2H), 6.33 (d, 1H), 5.39 (s,2H), 4.57 (s, 2H), 4.33 (s, 3H), 3.96 (s, 2H), 3.82 (s, 3H). E7-101

LC-MS: m/z 438 (M + H)⁺. ¹H NMR (400 MHz, DMSO-d6) δ: 12.78 (s, 1H),8.46 (s, 1H), 7.72 (s, 1H), 7.44 (s, 1H), 7.25 (s, 1H), 6.80 (s, 1H),6.26 (d, 1H), 5.26 (s, 2H), 4.48 (s, 2H), 4.26 (s, 3H), 3.70 (s, 3H),3.31 (s, 2H). E7-102

LC-MS: m/z 410 (M + H)⁺. ¹H NMR (400 MHz, DMSO-d6) δ: 12.79 (s, 1H),12.10 (s, 1H), 8.51 (s, 1H), 7.72 (s, 1H), 7.53 (s, 1H), 7.29 (s, 1H),7.08 (s, 1H), 6.27 (d, 1H), 5.72 (s, 2H), 4.50 (s, 2H), 4.30 (s, 3H)E7-103

LC-MS: m/z 402 (M + H)⁺. ¹H NMR (400 MHz, DMSO-d6) δ: 12.85 (s, 1H),8.59 (s, 1H), 7.80 (d, 1H), 7.14 (s, 1H), 6.33 (d, 1H), 5.36 (d, 2H),4.53 (d, 2H), 4.33 (s, 3H) E7-104

LC-MS: m/z 382 (M + H)⁺. ¹H NMR (400 MHz, DMSO-d6) δ: 12.80 (s, 1H),8.53 (s, 1H), 7.69 (s, 1H), 7.16 (d, 1H), 6.30 (s, 2H), 6.27 (s, 1H),5.88 (s, 1H), 5.41 (s, 2H), 4.50 (s, 2H), 4.27 (s, 3H). E7-105

LC-MS: m/z 384 (M + H)⁺. ¹H NMR (400 MHz, DMSO-d6) δ: 12.79 (s, 1H),11.31 (s, 1H), 8.54 (d, 1H), 7.69 (s, 1H), 6.27 (d, 1H), 5.17 (d, 2H),4.51 (s, 2H), 4.27 (s, 3H). E7-106

LC-MS: m/z 411 (M + H)⁺. ¹H NMR (400 MHz, DMSO-d6) δ: 12.78 (s, 1H),8.52 (s, 1H), 8.25-8.12 (m, 2H), 7.84 (s, 1H), 7.70 (s, 1H), 6.26 (d,1H), 5.30 (s, 2H), 4.48 (s, 2H), 4.26 (s, 3H) E7-107

LC-MS: m/z 383 (M + H)⁺. ¹H NMR (400 MHz, DMSO-d6) δ: 12.79 (s, 1H),8.51 (s, 1H), 8.36 (s, 2H), 7.67 (s, 1H), 6.26 (d, 1H), 5.31 (s, 1H),5.19 (s, 2H), 4.49 (s, 2H), 4.27 (s, 3H). E7-108

LC-MS: m/z 382 (M + H)⁺. ¹H NMR (400 MHz, DMSO-d6) δ: 12.79 (s, 1H),11.47 (s, 1H), 8.52 (s, 1H), 7.68 (s, 1H), 7.04 (s, 1H), 6.27 (s, 1H),5.17 (s, 2H), 4.50 (s, 2H), 4.28 (s, 3H) E7-109

LC-MS: m/z 394 (M + H)⁺. ¹H NMR (400 MHz, DMSO-d6) δ: 12.78 (s, 1H),8.56 (s, 1H), 8.11 (d, 1H), 7.71 (s, 1H), 6.60 (s, 2H), 6.27 (d, 1H),6.18 (d, 1H), 5.19 (s,2H), 4.50 (s, 2H), 4.26 (s, 3H) E7-110

LC-MS: m/z 394 (M + H)⁺. ¹H NMR (400 MHz, DMSO-d6) δ: 12.84 (s, 1H),8.55 (s, 1H), 7.99 (d, 1H), 7.77 (s, 1H), 6.86 (s, 2H), 6.44-6.16 (m,2H), 5.28 (s, 2H), 4.57 (s, 2H), 4.32 (s, 3H)

Example 8. Synthesis of Compounds E8-v, E8-vi, and ES-viii

Compound E8-i can be converted to intermediate E8-ii through eitherakylation or Mitsunobu reaction similar to Example E-iii to -vi (see (i)in Scheme E8). Oxidation of 8-ii with either mCPBA or oxone generatecompounds E8-v and/or E8-vi. Both compounds of I, OMs, OTs); Compound E-and/or E8-i can be converted to intermediate E8-vii through nucleophilicaromatic substitution reaction with compound E8-xi, using a base such asLiHMDS or t-BuOK. Compound E8-viii can be synthesized from compoundE8-vii using either SmI₂, or Zn in AcOH, or TES in ACl₃. As used herein,Ar1 and Ar2 are each independently optionally substituted 5-membered or6-membered heteroaryl.

Example 8A. Synthesis of2-((1H-pyrazol-3-yl)methyl)-6-((6-aminopyridin-2-yl)methyl)-4-methyl-4H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazin-5(6H)-oneand6-((6-aminopyridin-2-yl)methyl)-4-methyl-2-(1H-pyrazole-3-carbonyl)-4H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one

Step A tert-butylN-[(tert-butoxy)carbonyl]-N-[6-({4-methanesulfonyl-7-methyl-9-oxo-3-thia-5,7,10,11-tetraazatricyclo[6.4.0.0{2,6}]-dodeca-1(8),2(6),4,11-tetraen-10-yl})methyl)pyridin-2-yl]carbamate

A mixture of4-methyl-2-(methylsulfonyl)-4H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one(7.5 g, 26.4 mmol) and K₃PO₄ (8.3 g, 39.3 mmol) in anhydrous MeCN (300mL) was stirred at 70° C. for 1 hr under N₂. Followed a solution oftert-butylN-[(tert-butoxy)carbonyl]-N-[6-(bromomethyl)pyridin-2-yl]carbamate (11.2g, 29.0 mmol) in MeCN (30 mL) was added. After stirred at 70° C. for 2.5hr under N₂, the reaction mixture was quenched with sat. NH₄Cl andextracted with EA (300 mL×3). The combined organic layers were washedwith water and brine, dried over Na₂SO₄, filtered and the organic phasewas concentrated. The crude product was purified by flash chromatography(silica gel, 0˜50% ethyl acetate in petroleum ether) to give tert-butylN-[(tert-butoxy)carbonyl]-N-[6-({4-methanesulfonyl-7-methyl-9-oxo-3-thia-5,7,10,11-tetraazatricyclo[6.4.0.0{2,6}]dodeca-1(8),2(6),4,11-tetraen-10-yl}methyl)pyridin-2-yl]carbamate(5.5 g). LC-MS (ESI) found: 591.1 (M+H)⁺.

Step B. tert-Butyl(6-((4-methyl-1-oxo-2-((phenylsulfonyl)(1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-3-yl)methyl)-4H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazin-6(5H)-yl)methyl)pyridin-2-yl)carbamate

To a stirred mixture of3-((phenylsulfonyl)methyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazole(11.9 g, 33.8 mmol) in anhydrous THF (200 mL) was added LiHMDS (50 mL, 1M in THF) at −40° C. under argon. After 10 min, the mixture was warmedup to 10° C. and stirred for 1 hr, then tert-butylN-[(tert-butoxy)carbonyl]-N-[6-({4-methanesulfonyl-7-methyl-9-oxo-3-thia-5,7,10,11-tetraazatricyclo[6.4.0.0{2,6}]dodeca-1(8),2(6),4,11-tetraen-10-yl}methyl)pyridin-2-yl]carbamate(9.1 g, 15.4 mmol in 35 mL THF) was added. The reaction was stirred at10° C. for another 30 min. The reaction mixture was poured into aq.NH₄Cl, extracted with EtOAc (200 mL×3). The combined organic layers werewashed with water and brine, dried over anhydrous Na₂SO₄ andconcentrated. The crude product was purified by flash chromatography(silica gel, 0˜50% ethyl acetate in petroleum ether) to give tert-butyl(6-((4-methyl-5-oxo-2-((phenylsulfonyl)(1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-3-yl)methyl)-4H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazin-6(5H)-yl)methyl)pyridin-2-yl)carbamate(6.6 g). LC-MS (ESI) found: 763.2 (M+H)⁺.

Step C. tert-Butyl(6-((4-methyl-5-oxo-2-((1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-3-yl)methyl)-4H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazin-6(5H)-yl)methyl)pyridin-2-yl)carbamate

A solution of tert-butyl(6-((4-methyl-5-oxo-2-((phenylsulfonyl)(1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-3-yl)methyl)-4H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazin-6(5H)-yl)methyl)pyridin-2-yl)carbamate(6.0 g, 7.86 mmol) in EtOH/AcOH (35 mL/50 mL) was heated to 50° C. withvigorously stirred in the presence of Zn (2.55 g, 117.9 mmol) for 40min. Additional zinc were added every 40 min (2.55 g, twice, monitor thereaction by TLC/LC-MS to avoid the by-product and over reduced product).The solution was filtered and the filter cake was washed with DCM. Thefiltrate was partly evaporated, neutralized with saturated NaHCO₃solution, dried over MgSO₄ and the solvent was removed under vacuum. Thecrude product was purified by flash chromatography (silica gel,DCM:MeOH=40:1) to give tert-butyl(6-((4-methyl-5-oxo-2-((1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-3-yl)methyl)-4H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazin-6(5H)-yl)methyl)pyridin-2-yl)carbamate(3.1 g). LC-MS (ESI) found: 623.3 (M+H)⁺.

Step D.2-((1H-pyrazol-3-yl)methyl)-6-((6-aminopyridin-2-yl)methyl)-4-methyl-4H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one

To a mixture of tert-butyl(6-((4-methyl-5-oxo-2-((1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-3-yl)methyl)-4H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazin-6(5H)-yl)methyl)pyridin-2-yl)carbamate(3.0 g, 4.8 mmol) in ethanol (30 mL) was added HCl (30 mL, 4 M indioxane). The reaction mixture was stirred at 80° C. for 40 min. Thereaction mixture was cooled down to r.t., filtered and the solid wascollected, suspended in water and neutralized with aqueous NaHCO₃ at 10°C. Filtered to give the desire compound2-((1H-pyrazol-3-yl)methyl)-6-((6-aminopyridin-2-yl)methyl)-4-methyl-4H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one(1.5 g). LC-MS (ESI) found: 393.2 (M+H)⁺. ¹HNMR (400 MHz, DMSO-d₆) δ12.78 (s, 1H), 8.53 (s, 1H), 7.72 (s, 1H), 7.25 (dd, 1H), 6.33-6.24 (m,2H), 6.08 (d, 1H), 5.90 (s, 2H), 5.19 (s, 2H), 4.49 (s, 2H), 4.26 (s,3H).

Step E. Synthesis of tert-butyl(6-((4-methyl-5-oxo-2-(1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazole-3-carbonyl)-4H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazin-6(5H)-yl)methyl)pyridin-2-yl)carbamate

To a solution of tert-butyl(6-((4-methyl-5-oxo-2-((1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-3-yl)methyl)-4H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazin-6(5H)-yl)methyl)pyridin-2-yl)carbamate(100 mg, 0.16 mmol) in DMF (2 mL) was added K₂CO₃ (88 mg, 0.64 mmol).The mixture was stirred at 70° C. for 8 hr. The mixture was poured intowater, the precipitate was collected by filtration and purified bypre-TLC (2% MeOH in DCM) to afford tert-butyl(6-((4-methyl-5-oxo-2-(1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazole-3-carbonyl)-4H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazin-6(5H)-yl)methyl)pyridin-2-yl)carbamate(20 mg). LC-MS (ESI): m/z 637 (M+H)⁺.

Step F. Synthesis of6-((6-aminopyridin-2-yl)methyl)-4-methyl-2-(1H-pyrazole-3-carbonyl)-4H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one

To a solution of tert-butyl(6-((4-methyl-5-oxo-2-(1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazole-3-carbonyl)-4H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazin-6(5H)-yl)methyl)pyridin-2-yl)carbamate(20 mg, 0.03 mmol) in EtOH (1 mL) was added HCl (1 mL, 4 mol/L indioxane). The mixture was stirred at 80° C. for 1 hr and cooled down.The precipitate was collected by filtration and neutralized with sat.NaHCO₃. washed with water and dried to afford 5 mg of6-((6-aminopyridin-2-yl)methyl)-4-methyl-2-(1H-pyrazole-3-carbonyl)-4H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one.LC-MS (ESI): m/z 407 (M+H)⁺. ¹H NMR (400 MHz, DMSO-d₆) δ: 8.75 (s, 1H),7.96 (s, 1H), 7.50 (s, 1H), 7.31-7.22 (m, 1H), 6.31 (d, 1H), 6.14 (d,1H), 5.91 (s, 2H), 5.23 (s, 2H), 4.38 (s, 3H).

Example 8B. Synthesis of6-((1H-pyrazol-3-yl)methyl)-2-((6-(dimethylamino)pyridin-2-yl)methyl)-4-methyl-4,6-dihydro-5H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazin-5-one

Step A. (6-(dimethylamino)pyridin-2-yl)methanol

To a solution of (6-chloropyridin-2-yl)methanol (500 mg, 2.67 mmol) indimethylamine in THF (35 mL) was added Pd(OAc)₂ (78 mg, 0.35 mmol),Xantphos (170 mg, 0.29 mmol) and t-BuONa (385 mg, 4.01 mmol). Thereaction mixture was stirred at 100° C. for 18 h. The reaction mixturewas filtered and concentrated under reduced pressure. The residue waspurified by flash chromatography (silica gel, 0˜35% ethyl acetate inpetroleum ether) to afford (6-(dimethylamino)pyridin-2-yl)methanol (180mg). LCMS: 153 (M+H)⁺.

Step B. 6-(chloromethyl)-N,N-dimethylpyridin-2-amine

To a stirred mixture of (6-(dimethylamino)pyridin-2-yl)methanol (170 mg,1.1 mmol) in DCM (10 mL) was added SOCl₂ (665 mg, 5.6 mmol) at 0° C. Thereaction mixture was stirred at r.t for 1 hr. The reaction mixture wasadjusted at pH=7-8 with aq. NaHCO₃. Then the mixture was extracted withDCM, washed with water and brine. The organic layer was dried overNa₂SO₄, concentrated under reduced pressure to afford6-(chloromethyl)-N,N-dimethylpyridin-2-amine (70 mg). LCMS: 171 (M+H)⁺.

Step C. N,N-dimethyl-6-((phenylsulfonyl)methyl)pyridin-2-amine

To a stirred mixture of 6-(chloromethyl)-N,N-dimethylpyridin-2-amine(500 mg, 2.94 mmol) in DMSO (10 mL) was added PhSO₂Na (1.44 g, 8.82mmol) at r.t. The mixture was stirred at r.t for 18 hr. The reactionmixture was poured into water and extracted with DCM. The mixture waswashed with water and the organic layer was concentrated under reducedpressure. The residue was purified by flash chromatography (silica gel,0˜20% ethyl acetate in petroleum ether) to affordN,N-dimethyl-6-((phenylsulfonyl)methyl) pyridine-2-amine (380 mg). LCMS:277 (M+H)⁺.

Step D.2-((6-(dimethylamino)pyridin-2-yl)(phenylsulfonyl)methyl)-4-methyl-6-((1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-3-yl)methyl)-4H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one

To a stirred mixture of4-methyl-2-(methylsulfonyl)-6-((1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-3-yl)methyl)-4H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one(180 mg, 0.36 mmol), which was synthesized similar to compound E8-1 inExample 8A, in dry THF (10 mL) was addedN,N-dimethyl-6-((phenylsulfonyl)methyl)pyridin-2-amine (120 mg, 0.44mmol) and t-BuOK (122 mg, 1.1 mmol) at 60° C. under N₂. The mixture wasstirred at 60° C. for 2 h under N₂. Then the mixture was poured into thewater and extracted with EtOAc, washed with water and brine. The organiclayer was dried over Na₂SO₄, concentrated under reduced pressure andpurified by Prep-TLC (PE:EtOAc=1:1.5) to afford2-((6-(dimethylamino)pyridin-2-yl)(phenylsulfonyl)methyl)-4-methyl-6-((1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-3-yl)methyl)-4H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one(50 mg). LCMS: 691 (M+H)⁺.

Step E.2-((6-(dimethylamino)pyridin-2-yl)methyl-6-((1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-3-yl)methyl)-4H-thiazolo[(5′,4′:4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one

To a mixture of2-((6-(dimethylamino)pyridin-2-yl)(phenylsulfonyl)methyl)-4-methyl-6-((1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-3-yl)methyl)-4H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one(50 mg, 0.07 mmol) in THF (5 mL) and MeOH (5 mL) at r.t under N₂ wasadded SmI₂ (5 mL, 0.1M in THF) at −40° C. The reaction mixture wasstirred at −40° C. for 10 min and then quenched with water. Thefollowing mixture was extracted with EtOAc twice. The combined organiclayers were washed with brine (30 mL), dried over anhydrous Na₂SO₄ andconcentrated under reduced pressure. The residue was purified byprep-TLC (PE/EtOAc=1/1.5) to give the desired product (10 mg). LCMS: m/z551 (M+H)⁺.

Step F.6-((1H-pyrazol-3-yl)methyl)-2-((6-(dimethylamino)pyridin-2-yl)methyl)-4-methyl-4H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one

A mixture of2-((6-(dimethylamino)pyridin-2-yl)methyl)-4-methyl-6-((1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-3-yl)methyl)-4H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one(10 mg, 0.018 mmol) in DCM/TFA (2 mL/2 mL) was stirred at r.t for 1hour. The reaction mixture was concentrated. The residue was purified byprep-HPLC to afford desired product (1.4 mg). LCMS: 421 (M+H)⁺. ¹H NMR(400 MHz, DMSO) δ 8.51 (s, 1H), 7.55 (s, 1H), 7.48 (dd, 1H), 6.64 (d,1H), 6.54 (d, 1H), 6.11 (d, 1H), 5.32 (s, 2H), 4.44 (s, 2H), 4.26 (s,3H), 3.05 (s, 6H). The following compounds were synthesized according toScheme E8 and Example 8C using the appropriate starting material.Standard protection and deprotection can be used when necessary.

Cpd No. Structure and chemical name Charaterization E8-7 

LCMS: 393 (M + H)⁺. ¹H-NMR (400 MHz, DMSO) δ 12.67 (s, 1H), 8.50 (s,1H), 7.62 (s, 1H), 7.46 (s, 1H), 6.60 (d, 1H), 6.45 (s, 1H), 6.11 (d,1H), 5.33 (s, 2H), 4.42 (s, 2H), 4.26 (s, 3H) E8-8 

LCMS: m/z 385 (M + H)⁺. 1H NMR (400 MHz, DMSO- d6) δ 8.60 (s, 1H), 7.89(s, 1H), 7.75 (d, 1H), 7.67 (d, 1H), 5.65 (s, 2H), 4.63 (s, 2H), 4.27(s, 3H). E8-9 

LCMS: m/z 368 (M + H)⁺. 1H NMR (400 MHz, DMSO- d6) δ 12.79 (s, 1H), 8.51(s, 1H), 8.29 (s, 1H), 8.00 (s, 1H), 7.68 (s, 1H), 6.26 (s, 1H), 5.26(s, 2H), 4.49 (s, 2H), 4.27 (s, 3H). E8-10

LCMS: m/z 410 (M + H)⁺. 1H NMR (400 MHz, DMSO- d6) δ 9.03 (d, 1H), 8.52(s, 1H), 7.42 (d, 1H), 7.36 (t, 1H), 6.55 (d, 1H), 6.35 (d, 1H), 5.98(s, 2H), 5.48 (s, 2H), 4.37 (s, 2H), 4.26 (s, 3H) E8-11

LC-MS: m/z 401 (M + H)⁺. 1H NMR (400 MHz, DMSO) δ 9.13 (d, 1H), 8.60 (s,1H), 7.75 (d, 1H), 7.72 (d, 1H), 7.67 (d, 1H), 5.65 (s, 2H), 4.71 (s,2H), 4.27 (s, 3H). E8-12

LC-MS: m/z 395 (M + H)⁺. 1H NMR (400 MHz, DMSO) δ 8.60 (s, 1H), 8.57(dd, 1H), 7.81 (td, 1H), 7.74 (d, 1H), 7.67 (d, 1H), 7.51 (d, 1H),7.36-7.30 (m, 1H), 5.65 (s, 2H), 4.68 (s, 2H), 4.26 (s, 3H) E8-13

LC-MS: m/z 401 (M + H)⁺. 1H NMR (400 MHz, DMSO) δ 9.13 (d, 1H), 9.03 (d,1H), 8.54 (s, 1H), 7.72 (d, 1H), 7.44-7.41 (m, 1H), 5.48 (s, 2H), 4.71(s, 2H), 4.27 (s, 3H). E8-14

LC-MS: m/z 385 (M + H)+. 1H NMR (400 MHz, DMSO) δ 9.03 (d, 1H), 8.54 (s,1H), 7.89 (s, 1H), 7.42 (d, 1H), 5.48 (s, 2H), 4.63 (s, 2H), 4.26 (s,3H). E8-15

LCMS: m/z 392 (M + H)⁺. 1H NMR (400 MHz, DMSO) δ 8.56 (d, 1H), 8.50 (s,1H), 7.81 (td, 1H), 7.55 (d, 1H), 7.50 (d, 1H), 7.32 (dd, 1H), 6.07 (d,1H), 5.26 (s, 2H), 4.67 (s, 2H), 4.26 (s, 3H), 3.76 (s, 3H). E8-16

LCMS: m/z 379 (M + H)+. 1H NMR (400 MHz, DMSO) δ 8.56 (d, 1H), 8.52 (s,1H), 7.84-7.78 (m, 1H), 7.71 (s, 1H), 7.50 (d, 1H), 7.36-7.28 (m, 1H),5.42 (s, 2H), 4.67 (s, 2H), 4.26 (s, 3H). E8-17

LCMS: m/z 378 (M + H)⁺. 1H NMR (400 MHz, DMSO- d6) δ 12.63 (s, 1H),8.57-8.55 (m, 1H), 8.51 (s, 1H), 7.81 (dd, 1H), 7.59 (s, 1H), 7.50 (d,1H), 7.34-7.30 (m, 1H), 6.11 (d, 1H), 5.32 (s, 2H), 4.67 (s, 2H), 4.26(s, 3H). E8-18

LC-MS: m/z 414 (M + H)+. 1H NMR (400 MHz, DMSO) δ 12.63 (s, 1H), 8.51(s, 1H), 7.61 (s, 1H), 7.56 (s, 1H), 6.11 (s, 1H), 6.06 (t, 1H), 5.32(s, 2H), 4.70 (d, 2H), 4.60 (s, 2H), 4.27 (s, 3H). E8-19

LC-MS: m/z 431 (M + 1)+. 1H NMR (400 MHz, DMSO) δ 8.60 (s, 1H), 7.75 (d,1H), 7.67 (d, 1H), 7.57 (s, 1H), 6.10-5.97 (m, 1H), 5.65 (s, 2H), 4.70(d, 2H), 4.61 (s, 2H), 4.26 (s, 3H). E8-20

LC-MS: m/z 428 (M + 1)⁺. 1H NMR (400 MHz, DMSO) δ 8.49 (s, 1H),7.62-7.58 (m, 2H), 6.07-6.03 (m, 2H), 5.26 (s, 2H), 4.70 (d, 2H), 4.60(s, 2H), 4.26 (s, 3H), 3.76 (s, 3H). E8-21

LC-MS: m/z 415 (M + 1)+. 1H NMR (400 MHz, DMSO) δ 14.77 (s, 1H), 8.52(s, 1H), 7.65 (s, 1H), 7.56 (s, 1H), 6.05 (t, 1H), 5.42 (s, 2H), 4.70(d, 2H), 4.60 (s, 2H), 4.26 (s, 3H). E8-22

LCMS: m/z 416 (M + H)+. 1H NMR (400 MHz, DMSO) δ 9.13 (d, 1H), 8.51 (s,1H), 7.72 (d, 1H), 7.03 (br s, 2H), 6.22 (s, 1H), 5.13 (s, 2H), 4.71 (s,2H), 4.27 (s, 3H). E8-23

LCMS: m/z 382 (M + H)+. 1H NMR (400 MHz, DMSO- d6) δ 8.50 (s, 1H), 7.89(s, 1H), 7.56 (d, 1H), 6.07 (d, 1H), 5.26 (s, 2H), 4.63 (s, 2H), 4.26(s, 3H), 3.76 (s, 3H). E8-24

LCMS: m/z 368 (M + H)+. 1H NMR (400 MHz, DMSO- d6) δ 14.73 (s, 1H),12.64 (s, 1H), 8.51 (s, 1H), 7.89 (s, 1H), 7.61 (s, 1H), 6.11 (d, 1H),5.33 (s, 2H), 4.63 (s, 2H), 4.26 (s, 3H). E8-25

LC-MS: m/z 384.0 (M + H)+. 1H NMR (400 MHz, DMSO) δ 12.62 (s, 1H), 9.12(d, 1H), 8.51 (s, 1H), 7.72 (d, 1H), 7.61 (s,lH), 6.11 (s, 1H), 5.32 (s,2H), 4.70 (s, 2H), 4.27 (s, 3H). E8-26

LC-MS: 440.0 [M + H]⁺ 1H NMR (400 MHz, DMSO) δ 8.55 (s, 1H), 7.57 (s,1H), 7.29-7.21 (m, 1H), 6.29 (d, 1H), 6.13-6.07 (m, 2H), 5.92 (s, 2H),5.19 (s, 2H), 4.70 (d, 2H), 4.61 (s, 2H), 4.26 (s, 3H). E8-27

LCMS: 418 (M + H)⁺. ¹H NMR (400 MHz, DMSO) δ 8.53 (s, 1H), 7.74 (s, 1H),6.92 (s, 2H), 5.43 (s, 2H), 4.27 (s, 2H), 4.26 (s, 3H). E8-28

LCMS: 403 (M + H)⁺. ¹H NMR (400 MHz, DMSO) δ 8.70 (d, 1H), 8.54 (s, 1H),7.73 (s, 1H), 5.42 (s, 2H), 4.60 (d, 2H), 4.25 (s, 3H). E8-29

LCMS: 429 (M + H)⁺. ¹H NMR (400 MHz, DMSO) δ 9.09 (d, 1H), 8.57 (s, 1H),7.88 (d, 1H), 7.73 (s, 1H), 6.12 (s, 1H), 5.54-5.34 (m, 2H), 4.23 (s,3H), 3.76-3.61 (m, 2H), 1.20 (q, 3H). E8-30

LCMS: m/z 398 (M + H)+. ¹H NMR (400 MHz, DMSO) δ 9.12 (d, 1H), 8.50 (s,1H). 7.72 (d, 1H), 7.56 (d, 1H), 6.07 (d, 1H), 5.26 (s, 2H), 4.70 (s,2H), 4.26 (s, 3H), 3.76 (s, 3H). E8-31

LCMS: m/z 400 (M + H)+. ¹H NMR (400 MHz, DMSO) δ 8.50 (s, 1H), 7.89 (s,1H), 6.90 (s, 2H), 6.18 (s, 1H), 5.02 (s, 2H), 4.63 (s, 2H) 4.26 (s,3H). E8-32

LCMS: m/z 419 (M + H)+. ¹H NMR (400 MHz, DMSO) δ 9.09 (s, 1H), 8.53 (s,1H), 7.73 (s, 1H), 5.42 (s, 2H), 4.65 (s, 2H), 4.25 (s, 3H). E8-33

LCMS: m/z 418 (M + H)+. ¹H NMR (400 MHz, DMSO) δ 9.09 (s, 1H), 8.51 (s,1H), 7.55 (s, 1H), 6.11 (d, 1H), 5.32 (s, 2H), 4.65 (s, 2H), 4.26 (s,3H). E8-34

LC-MS m/z 386.0 (M + H)⁺. ¹H NMR (400 MHz, DMSO) δ 14.82 (s, 1H), 12.82(s, 1H), 8.57 (s, 1H), 7.95 (s, 1H), 7.70 (s, 1H), 5.47 (s, 2H), 4.54(s, 2H), 4.31 (s, 3H) E8-35

LCMS: m/z 462 (M + H)⁺. ¹H NMR (400 MHz, DMSO- d₆) δ 12.64 (s, 1H), 9.19(s, 1H), 8.51 (s, 1H), 7.57 (s, 1H), 6.11 (d, 1H), 5.33 (s, 2H), 4.65(s, 2H), 4.26 (s, 3H). E8-36

LCMS: m/z 394 (M + H)+. ¹H NMR (400 MHz, DMSO) δ 12.77 (s, 1H), 11.71(s, 1H), 8.58 (s, 1H), 7.70 (s, 1H), 7.32 (s, 1H), 6.25-6.05 (m, 2H),5.75 (s, 1H), 5.19 (s, 2H), 4.50 (s, 2H), 4.26 (s, 3H). E8-37

LCMS: m/z 417 (M + H)⁺. ¹H NMR DMSO-d6 400 MHz δ 12.65 (s, 1H), 8.51 (s,1H), 7.57 (s, 1H), 6.92 (s, 2H), 6.12 (d, 1H), 5.33 (s, 2H), 4.26 (s,5H). E8-38

LCMS: m/z 402 (M + H)⁺. ¹H NMR DMSO-d6 400 MHz δ 12.70 (s, 1H), 8.70 (d,1H), 8.52 (s, 1H), 7.55 (s, 1H), 6.11 (d, 1H), 5.33 (s, 2H), 4.60 (d,2H), 4.26 (s, 3H). E8-39

LCMS: m/z 389 (M + H)⁺. ¹H NMR DMSO-d6 400 MHz δ 8.61-8.54 (m, 2H),8.51- 8.46 (m, 1H), 7.82 (td, 1H), 7.73 (td, 1H), 7.52 (d, 1H), 7.34(ddd, 1H), 7.27 (dd, 1H), 7.14 (d, 1H), 5.46 (s, 2H), 4.69 (s, 2H), 4.26(s, 3H). E8-40

LCMS: m/z 385 (M + H)⁺. ¹H NMR (400 MHz, DMSO- d₆) δ 9.13 (d, 1H), 8.53(s, 1H), 7.74 (s, 1H), 7.72 (d, 1H), 5.42 (s, 2H), 4.70 (s, 2H), 4.26(s, 3H). E8-41

LCMS: m/z 381 (M + H)⁺. ¹H NMR (400 MHz, DMSO- d₆) δ 12.77 (s, 1H), 8.48(s, 1H), 7.71 (s, 1H), 7.55 (d, 1H), 6.26 (d, 1H), 6.07 (d, 1H), 5.26(s, 2H), 4.47 (s, 2H), 4.27 (s, 3H), 3.76 (s, 3H). E8-42

LCMS: m/z 407 (M + H)⁺. ¹H NMR (400 MHz, DMSO- d₆) δ 8.48 (s, 1H), 7.55(d, 1H), 7.35 (t, 1H), 6.55 (d, 1H), 6.35 (d, 1H), 6.07 (d, 1H), 5.97(s, 2H), 5.26 (s, 2H), 4.36 (s, 2H), 4.26 (s, 3H), 3.76 (s, 3H). E8-43

LCMS: m/z 382 (M + H)⁺. ¹H NMR (400 MHz, DMSO- d₆) δ 14.70 (s, 1H), 8.51(s, 1H), 7.74 (s, 1H), 7.65 (d, 1H), 6.22 (d, 1H), 5.42 (s, 2H), 4.43(s, 2H), 4.26 (s, 3H), 3.81 (s, 3H). E8-44

LCMS: m/z 395 (M + H)⁺. ¹H NMR (400 MHz, DMSO- d₆) δ 8.48 (s, 1H), 7.65(d, 1H), 7.55 (d, 1H), 6.22 (d, 1H), 6.07 (d, 1H), 5.26 (s, 2H), 4.43(s, 2H), 4.26 (s, 3H), 3.82 (s, 3H), 3.76 (s, 3H). E8-45

LCMS: m/z 403 (M + H)⁺. ¹H NMR (400 MHz, DMSO- d₆) δ 13.02-12.57 (m,2H), 8.49 (s, 1H), 7.6-7.8 (m, 2H), 5.33 (s, 2H), 4.49 (s, 2H), 4.27 (s,3H). E8-46

LCMS: m/z 410 (M + H)⁺. ¹H NMR (400 MHz, DMSO- d₆) δ 9.13 (d, 1H), 8.55(s, 1H), 7.72 (d, 1H), 7.25 (t, 1H), 6.29 (d, 1H), 6.07 (d, 1H), 5.92(s, 2H), 5.19 (s, 2H), 4.71 (s, 2H), 4.26 (s, 3H). E8-47

LCMS: m/z 385 (M + H)⁺. ¹H NMR (400 MHz, DMSO) δ 12.77 (s, 1H), 12.62(s, 1H), 8.50 (s, 1H), 7.87 (d, 1H), 7.61 (s, 1H), 6.11 (s, 1H), 5.32(s, 2H), 4.49 (s, 2H), 4.26 (s, 3H). E8-48

LCMS: m/z 399 (M + H)+. ¹H NMR (400 MHz, DMSO- d6) δ 8.95 (s, 1H), 8.55(s, 1H), 7.79 (s, 1H), 5.47 (s, 2H), 4.66 (s, 2H), 4.31 (s, 3H), 2.55(s, 3H). E8-49

LCMS: m/z 381 (M + H)⁺. ¹H NMR (400 MHz, DMSO- d6) δ 12.64 (s, 1H), 8.49(s, 1H), 7.65 (s, 1H), 7.56 (s, 1H), 6.23 (d, 1H), 6.11 (d, 1H), 5.32(s, 2H), 4.43 (s, 2H), 4.26 (s, 3H), 3.82 (s, 2H). E8-50

LCMS: m/z 398 (M + H)⁺. ¹H NMR (400 MHz, DMSO- d6) δ 12.63 (s, 1H), 8.90(s, 1H), 8.48 (s, 1H), 7.60 (s, 1H), 6.11 (d, 1H), 5.32 (s, 2H), 4.61(s, 2H), 4.26 (s, 3H) 2.50 (s, 3H overlap with DMSO-d6). . E8-51

LCMS: m/z 399 (M + H)⁺. ¹H NMR (400 MHz, DMSO- d6) δ 12.83 (s, 1H), 8.54(s, 1H), 7.91 (s, 1H), 7.61 (s, 1H), 6.13 (s, 1H), 5.32 (s, 2H), 4.54(s, 2H), 4.34 (s, 3H), 3.81 (s, 3H). E8-52

LCMS: m/z 425 (M + H)⁺. ¹H NMR (400 MHz, DMSO) δ 12.68 (s, 1H), 8.56 (s,1H), 7.65 (s, 1H), 7.43 (t, 1H), 6.62 (d, 1H), 6.46 (dd, 1H), 6.16 (s,1H), 5.37 (s, 2H), 4.52 (s, 2H), 4.31 (s, 3H), 2.83 (d, 3H) E8-53

LC-MS: m/z 411 (M + H)⁺. ¹H NMR (400 MHz, DMSO- d₆) δ 12.64 (s, 1H),8.50 (s, 1H), 7.57 (s, 1H), 7.36 (t, 1H), 6.41 (dd, 1H), 6.11 (d, 1H),5.96 (s, 2H), 5.32 (s, 2H), 4.44 (d, 2H), 4.26 (s, 3H) E8-54

LC-MS: m/z 410 (M + H)⁺. ¹H NMR (400 MHz, DMSO- d₆) δ 8.52 (s, 1H), 8.49(s, 1H), 7.83 (d, 1H), 7.51 (d, 1H), 7.40-7.25 (m, 1H), 6.91 (s, 2H),6.17 (s, 1H), 5.12 (s, 2H), 4.67 (s, 2H), 4.26 (s, 3H) E8-55

LC-MS: m/z 395 (M + H)⁺. ¹H NMR (400 MHz, DMSO- d₆) δ 9.09 (d, 1H), 8.63(m, 1H), 8.59 (s, 1H), 7.88 (m, 1H), 7.57 (d 1H), 7.51 (s, 1H), 7.39(dd, 1H), 5.54 (s, 2H), 4.73 (s, 2H), 4.32 (s, 3H) E8-56

LC-MS: m/z 369 (M + H)⁺. ¹H NMR (400 MHz, DMSO- d₆) δ 8.53 (s, 1H), 7.89(s, 1H), 7.74 (s 1H), 5.43 (s, 2H), 4.63 (s, 2H), 4.26 (s, 3H) E8-57

LC-MS: m/z 404 (M + H)⁺. ¹H NMR (400 MHz, DMSO- d₆) δ 8.57 (s, 1H), 8.54(s, 1H), 7.82 (dd, 1H), 7.51 (d 1H), 7.33 (dd, 1H), 7.27-7.22 (m, 1H),6.29 (d 1H), 6.07 (d 1H), 5.90 (s, 2H), 5.19 (s, 2H), 4.68 (s, 2H), 4.26(s, 3H) E8-58

LC-MS: m/z 394 (M + H)⁺. ¹H NMR (400 MHz, DMSO- d₆) δ 8.55 (s, 1H), 7.90(s, 1H), 7.26 (dd 1H), 6.30 (d, 1H), 6.07 (d 1H), 5.91 (s, 2H), 5.19 (s,2H), 4.64 (s, 2H), 4.26 (s, 3H) E8-59

LC-MS: m/z 396 (M + H)⁺. ¹H NMR (400 MHz, DMSO- d₆) δ 12.60 (s, 1H),8.57 (s, 1H), 8.55 (s, 1H), 7.90-7.80 (m, 1H), 7.68 (s, 1H), 7.49 (d1H), 7.31 (dd, 1H), 5.32 (s, 2H), 4.65 (s, 2H), 4.24 (s, 3H) E8-60

LC-MS: m/z 412 (M + H)⁺. ¹H NMR (400 MHz, DMSO- d₆) δ 13.00 (s, 1H),8.67 (d, 1H), 8.46 (s, 1H), 7.86 (s, 1H), 7.82 (dd, 1H), 7.51 (d, 1H),7.33 (dd, 1H), 5.34 (s, 2H), 4.67 (s, 2H), 4.26 (s, 3H E8-61

LC-MS: m/z 492 (M + H)⁺. ¹H NMR (400 MHz, DMSO- d6) δ: 12.64 (s, 1H),8.52 (s, 1H), 7.61 (s, 1H), 6.11 (s, 1H), 5.32 (s, 2H), 4.44 (s, 2H),4.26 (s, 3H), 3.99 (s, 3H). E8-62

LC-MS: m/z 414 (M + H)⁺. ¹H NMR (400 MHz, DMSO- d6) δ: 12.63 (s, 1H),8.57 (s, 1H), 8.50 (s, 1H), 7.60 (s, 1H), 6.11 (d, 1H), 5.32 (s, 2H),4.47 (s, 2H), 4.26 (s, 3H), 3.97 (s, 3H). E8-63

LC-MS: m/z 425 (M + H)⁺. ¹H NMR (400 MHz, DMSO- d6) δ: 9.12 (s, 1H),8.19 (s, 1H), 7.66 (s, 1H), 6.25 (s, 1H), 5.59 (s, 2H), 4.49 (s, 2H),4.31 (s, 3H), 3.81 (s, 3H). E8-64

LC-MS: m/z 419 (M + H)⁺. ¹H NMR (400 MHz, DMSO- d6) δ: 13.28-12.57 (m,2H), 8.49 (s, 1H), 7.90-7.50 (m, 2H), 5.34 (s, 2H), 4.50 (s, 2H), 4.26(s, 3H). E8-65

LC-MS: m/z 428 (M + H)⁺. ¹H NMR (400 MHz, DMSO- d6) δ: 12.89-12.76 (m,2H), 8.49 (s, 1H), 8.22 (s, 1H), 7.86 (s, 1H), 7.60 (s, 1H), 7.00 (s,1H), 5.57 (s, 2H), 4.51 (s, 2H), 4.26 (s, 3H). E8-66

LC-MS: m/z 426 (M + H)⁺. ¹H NMR (400 MHz, DMSO- d6) δ: 12.80 (s, 1H),8.57 (d, 1H), 8.08 (dd, 1H), 7.92 (d, 1H), 7.16 (d, 1H), 6.90 (dd, 1H),5.29 (s, 2H), 4.50 (s, 2H), 4.26 (s, 3H), 3.92 (s, 3H). E8-67

LC-MS: m/z 412 (M + H)⁺. ¹H NMR (400 MHz, DMSO- d6) δ: 12.78 (s, 1H),11.71 (s, 1H), 8.56 (s, 1H), 7.87 (s, 1H), 7.30 (d, 1H), 6.82 (d, 1H),6.08 (dd, 1H), 5.10 (s, 2H), 4.50 (s, 2H), 4.26 (s, 3H). E8-68

LC-MS: m/z 410 (M + H)⁺. ¹H NMR (400 MHz, DMSO- d6) δ: 13.63 (s, 1H),12.81 (s, 1H), 8.55 (s, 1H), 8.37 (s, 1H), 7.81 (s, 1H), 5.45 (s, 2H),4.51 (s, 2H), 4.25 (s, 3H). E8-69

LC-MS: m/z 467 (M + H)⁺. ¹H NMR (400 MHz, DMSO- d6) δ: δ10.35 (s, 1H),8.49 (s, 1H), 8.00 (s, 1H), 7.70 (dd, 2H), 7.45 (s, 1H), 5.45 (s, 2H),4.34 (s, 2H), 4.26 (s, 3H), 3.83 (s, 3H), 2.01 (s, 3H) E8-70

LC-MS: m/z 395 (M + H)⁺. ¹H NMR (400 MHz, DMSO- d6) δ 13.85 (s, 1H),8.70 (s, 1H), 8.04 (s, 1H), 7.58 (d, 1H), 7.38 (s, 1H), 6.12 (d, 1H),5.30 (s, 2H), 4.38 (s, 3H), 3.77 (s, 3H).

Example 9. Synthesis of Compounds E9-vi and E9-vii

Compound E9-iv can be synthesized by two approaches, (i) and (ii) inScheme 9. For approach (i), compound E9-ii can be synthesized fromcompound E9-i through alkylation reaction as showed in Example 7 orExample 8. As used herein, X^(a) is a leaving group. Formylationreaction of compound E9-ii with LiHMDS and DMF provides intermediateE9-iii. E9-iii reacts with a reducing agent (e.g. NaBH₄) to providecompound E9-iv. Alternatively in approach (ii), halogenation of compoundE9-i generates compound E9-ix. Compound E9-ix undergoes Stille reaction,ozonolysis, and reduction to furnish compound E9-x. Compound E9-x can bealkylated with E9-viii to provide compound E9-iv. In Scheme 9, (iii),Compound E9-iv undergoes halogenation to give intermediate E9-v (X^(b)is halogen such as Cl or Br). A metal (e.g. Pd or Cu) catalyzed couplingof E9-v with organic Tin, boron, zinc or magnesium provides compoundE9-vi. As used herein, M is an organic metal complex (e.g. organoboroncomplex such as boronic acid or pinaco boron complex, organotin complexsuch as —Sn(Bu^(t))₃; organozinc complex such as —Zn(halogen)); CompoundE9-v can also react with some nucleophiles such as nitrogen in aheterocycle to give product E9-vii. As used herein, Ar₁ and Ar₂ are eachindependently optionally substituted 5-membered or 6-memberedheteroaryl, X^(a) is a leaving group (e.g. Br, I, OMs, or OTs); andX^(b) is halogen.

Example 9A. Synthesis of6-((1H-indazol-4-yl)methyl)-4-methyl-2-(thiazol-4-ylmethyl)-4H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one

Step A. Synthesis of4-methyl-5-oxo-6-((1-((2-(trimethylsilyl)ethoxy)methyl)-1H-indazol-4-yl)methyl)-5,6-dihydro-4H-triazolo[5′,4:4,5]pyrrolo[2,3-d]pyridazine-2-carbaldehyde

To a mixture of4-methyl-6-((1-((2-(trimethylsilyl)ethoxy)methyl)-1H-indazol-4-yl)methyl)-4H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one(2.6 g, 5.57 mmol, 1 eq) in dry THF (30 mL) was added LiHMDS (1 M, 11.14mL, 2.0 eq) at −78° C.. The mixture was stirred at −78° C. for 2 hr.Then DMF (2.04 g, 27.86 mmol, 2.14 mL, 5.0 eq) was added dropwise to theabove mixture. The mixture was stirred at −78° C. for 2 hr. TLC(PE:EA=2:1, UV=254 nm) showed that one main new spot was formed. Themixture was poured into cold sat. NH₄Cl (20 mL). Then the mixture waswarmed to room temperature. The mixture was extracted with EtOAc (40mL×3). The organic layer was washed by water (20 mL×3) and concentratedin vacuo to give the desired product (2.6 g, crude). LCMS: m/z 495.2[M+H]⁺

Step B. Synthesis of2-(hydroxymethyl)-4-methyl-6-((1-((2-(trimethylsilyl)ethoxy)methyl)-1H-indazol-4-yl)methyl)-4H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one

To a mixture of crude 4-methyl-5-oxo-6-((1-((2-(trimethylsilyl)ethoxy)methyl)-1H-indazol-4-yl)methyl)-5,6-dihydro-4H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazine-2-carbaldehyde(1.0 g, 2.02 mmol, 1 eq) in THF (10 mL) and MeOH (10 mL) was added NaBH₄(152.97 mg, 4.04 mmol, 2 eq). The mixture was stirred at 30° C. for 14hr. TLC (DCM:MeOH=10:1, UV=254 nm) showed that the starting material wasconsumed completely and one main new spot was formed. The reaction wasquenched by addition of water (20 mL) and extracted with EtOAc (30mL×3). The combined organic layers were washed with brine (20 mL). Theorganic phase was concentrated in vacuo. The residue was purified byflash silica gel chromatography (ISCO®; 40 g SepaFlash® Silica FlashColumn, Eluent of 0-5% MeOH/DCM @ 30 mL/min). The eluent wasconcentrated in vacuo to give the desired product (382 mg). LCMS: m/z497.1 [M+H]⁺. ¹H NMR (400 MHz, DMSO-d₆) δ ppm 8.61 (s, 1H), 8.25 (s,1H), 7.66 (d, 1H), 7.38 (t, 1H), 7.05 (d, 1H), 6.36 (t, 1H), 5.74 (s,2H), 5.68 (s, 2H), 4.89 (d, 2H), 4.26 (s, 3H), 3.50 (t, 2H), 0.78 (t,2H), −0.12 (s, 9H).

Step C. Synthesis of2-(chloromethyl)-4-methyl-6-((1-((2-(trimethylsilyl)ethoxy)methyl)-1H-indazol-4-yl)methyl)-4H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one

To a mixture of 2-(hydroxymethyl)-4-methyl-6-((1-((2-(trimethylsilyl)ethoxy)methyl)-1H-indazol-4-yl)methyl)-4H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one(150.0 mg, 302.02 umol, 1 eq) and Et₃N (61.12 mg, 604.04 umol, 84.08 uL,2.0 eq) in DCM (5 mL) was added 4-methylbenzenesulfonyl chloride (75.0mg, 393.40 umol, 1.30 eq). The mixture was stirred at 30° C. for 5 hr.TLC (PE:EA=4:1, UV=254 nm) showed the starting material was consumedcompletely. Water (10 mL) and DCM (20 mL) was added to the mixture. Theorganic layers were concentrated in vacuo to give a yellow gum (0.1 g).The residue was purified by flash silica gel chromatography (ISCO®; 4 gSepaFlash® Silica Flash Column, Eluent of 0-20% Ethyl acetate/Petroleumether gradient @ 30 mL/min). The desired fraction was concentrated invacuo to give the desired product (40.0 mg, 76.88 umol). LCMS: m/z 515.1[M+H]+. ¹H NMR (400 MHz, CHLOROFORM-d₆) δ ppm 8.27 (d, 1H), 8.26 (s,1H), 7.52 (d, 1H), 7.39 (dd, 1H), 7.25 (s, 1H), 5.76 (s, 2H), 5.72 (s,2H), 4.96 (s, 2H), 4.40 (s, 3H), 3.50-3.57 (m, 2H), 0.84-0.90 (m, 2H),−0.09 to −0.06 (m, 9H).

Step D. Synthesis of4-methyl-2-(thiazol-4-ylmethyl)-6-((1-((2-(trimethylsilyl)ethoxy)methyl)-1H-indazol-4-yl)methyl)-4,6-dihydro-5H-thiazolo[5′,4′:4;5]pyrrolo[2,3-d]pyridazin-5-one

To a solution of2-(chloromethyl)-4-methyl-6-((1-((2-(trimethylsilyl)ethoxy)methyl)-1H-indazol-4-yl)methyl)-4H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one(50 mg, 0.97 mmol) and 4-(tributylstannyl)thiazole (114 mg, 2.91 mmol)in toluene (4 mL) was added Pd(PPh₃)₄(402 mg, 2.91 mmol). Then themixture was heated in MW reactor at 120° C. for 30 min under N₂. Thesolution was poured into water and extracted with EtOAc, dried overanhydrous Na₂SO₄. The organic layer was concentrated under reducedpressure. The residue was purified by flash chromatography (silica gel,0˜50% ethyl acetate in petroleum ether) to give4-methyl-2-(thiazol-4-ylmethyl)-6-((1-((2-(trimethylsilyl)ethoxy)methyl)-1H-indazol-4-yl)methyl)-4H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one(30 mg). LCMS: 564 (M+H)⁺.

Step E. Synthesis of6-((1H-indazol-4-yl)methyl)-4-methyl-2-(thiazol-4-ylmethyl)-4,6-dihydro-5H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazin-5-one

To a mixture of compound E9-4 (30 mg, 0.05 mmol) in DCM (3 mL) at r.t.under N₂ was added TFA (3 mL). The reaction mixture was stirred at r.t.for 1 h. The mixture was concentrated under reduced pressure. Theresidue was purified by prep-HPLC (C18, 0˜90% acetonitrile in H₂O with0.1% formic acid) to give the desired product (3.9 mg). LCMS: 434(M+H)⁺. ¹H NMR (400 MHz, DMSO) δ 13.12 (s, 1H), 9.12 (d, 1H), 8.56 (s,1H), 8.14 (s, 1H), 7.71 (d, 1H), 7.45 (d, 1H), 7.35-7.24 (m, 1H), 6.96(d, 1H), 5.65 (s, 2H), 4.70 (s, 2H), 4.27 (s, 3H).

Example 9B. Synthesis of4-methyl-2-(pyridin-2-ylmethyl)-6-(pyridin-3-ylmethyl)-4H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one

Step A. Synthesis of2-(hydroxymethyl)-4-methyl-6-(pyridin-3-ylmethyl)-4H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one

At −78° C., to a mixture of4-methyl-6-(pyridin-3-ylmethyl)-4H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one(640 mg, 2.15 mmol) in THF (10 mL) was added LiHMDS (4.3 mL, 1 M inTHF). After 30 min, dry DMF (0.84 mL, 10.8 mmol) was added to themixture. After the completely consumption of starting material, amixture of NaBH₄ (164 mg, 4.3 mmol) in EtOH (4 mL) was added and stirredfor 5 min. Then the mixture was poured into satd. NH₄Cl, extracted withEtOAc. The organic layer was washed with brine, dried over anhy. Na₂SO₄,filtered and concentrated. The residue was purified by flashchromatography (silica gel, 0˜5% MeOH in DCM) to afford2-(hydroxymethyl)-4-methyl-6-(pyridin-3-ylmethyl)-4H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one(220 mg). LC-MS (ESI): m/z 328 (M+H)⁺.

Step B. Synthesis of2-(chloromethyl)-4-methyl-6-(pyridin-3-ylmethyl)-4H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one

To a mixture of2-(hydroxymethyl)-4-methyl-6-(pyridin-3-ylmethyl)-4H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one(100 mg, 0.31 mmol) in DCM (5 mL) were added Et₃N (0.43 mL, 3.1 mmol)and MsCl (0.12 mL, 1.5 mmol). The reaction was stirred at roomtemperature for 6 hr. Then the mixture was washed with satd. NH₄Cl(aq.), dried over anhy. Na₂SO₄, filtered and concentrated. The residuewas purified by flash chromatography (silica gel, 0˜50% EtOAc in PE) toafford 65 mg of2-(chloromethyl)-4-methyl-6-(pyridin-3-ylmethyl)-4H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one.LC-MS (ESI): m/z 346 (M+H)⁺.

Step C. Synthesis of4-methyl-2-(pyridin-2-ylmethyl)-6-(pyridin-3-ylmethyl)-4H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one

Under nitrogen, to a mixture of2-(chloromethyl)-4-methyl-6-(pyridin-3-ylmethyl)-4H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one(50 mg, 0.14 mmol) and 2-(tributylstannyl)pyridine (0.14 mL, 0.43 mmol)in toluene (3 mL) was added Pd(PPh₃)₄(17 mg, 0.014 mmol). The reactionmixture was stirred at 100° C. overnight. Then the mixture was cooled,concentrated under reduced pressure and the residue was purified byprep-TLC (eluant: 10% MeOH in DCM) to afford 2 mg of4-methyl-2-(pyridin-2-ylmethyl)-6-(pyridin-3-ylmethyl)-4H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one.LC-MS (ESI): m/z 389 (M+H)⁺. ¹H NMR (400 MHz, DMSO-d₆) δ 8.56 (m, 3H),8.48 (dd, 1H), 7.81 (td, 1H), 7.71 (d, 1H), 7.50 (d, 1H), 7.37-7.30 (m,2H), 5.38 (s, 2H), 4.67 (s, 2H), 4.25 (s, 3H).

Example 9C. Synthesis of2-((1H-1,2,4-triazol-1-yl)methyl)-6-((1H-pyrazol-3-yl)methyl)-4-methyl-4H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazin-5(6H)-oneand6-((1H-pyrazol-3-yl)methyl)-2-((4H-1,2,4-triazol-4-yl)methyl)-4-methyl-4H-thiazolo[5′,4:4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one

Step A. Synthesis of1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-3-carbaldehyde

To a suspension of 1H-pyrazole-3-carbabaldehyde (10.0 g, 104.07 mol, 1eq) and DIPEA (33.63 g, 260.18 mmol, 45.32 m, 2.5 eq) in DCM (500 mL)was added dropwise 2-(chloromethoxy)ethyl-trimethyl-silane (26.03 g,156.11 mmol, 27.63 mL, 1.5 eq) at −40° C. Then the reaction mixture waswarmed to room temperature and stirred for 16 hr. TLC (petroleumether:EtOAc=5:1) showed the starting material was consumed completely,and two new spots were formed. The reaction mixture was concentrated invacuo. The residue was combined with another 2 batches (10.0 g each) andpurified by Combiflash (from 100% of petroleum ether to 40% of EtOAc inpetroleum ether) to give desired product 60.0 g. (note: mixture of 2regioisomers with ˜5/4 ratio). ¹H NMR (400 MHz, CHLOROFORM-d) δ ppm10.06 (s, 1H), 10.00 (s, 1H), 7.68 (d, 1H), 7.67 (d, 1H), 7.03 (d, 1H),6.92 (d, 1H), 5.87 (s, 2H), 5.56 (s, 2H), 3.61-3.67 (i, 4H), 0.91-1.01(m, 4H), −0.09-0.05 (m, 18H).

Step B. Synthesis of(1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-3-yl)methanol

To a solution of1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazole-3-carbaldehyde (30 g,132.54 mmol, 1 eq, mixture of 2 regioisomers with ˜5/4 ratio) in THF(200 mL)/MeOH (100 mL) was added NaBH₄ (7.52 g, 198.81 mmol, 1.50 eq) inportions at 0° C., the reaction mixture was stirred at 0° C. to roomtemperature for 18 hr. TLC (petroleum ether:EtOAc=2:1) showed thestarting materials were consumed completely, and two new spots wereformed. The solvent was concentrated in vacuo. The residue was purifiedby Combiflash (100% of petroleum ether to 100% of EtOAc) to give 25 g ofthe desired product. (Note: mixture of 2 regioisomers ratio ˜3/2). ¹HNMR (400 MHz, CHLOROFORM-d) δ ppm 7.55 (d, 1H), 7.47 (brs, 1H), 6.36 (d,1H), 6.34 (d, 1H), 5.57 (s, 2H), 5.42 (s, 2H), 4.74-4.76 (m, 4H),3.55-3.60 (m, 4H), 0.85-0.96 (m, 4H), 0.00-0.06 (m, 18H).

Step C. Synthesis of3-(bromomethyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazole

To a solution of(1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-3-yl)methanol (23 g,100.72 mmol, 1 eq, mixture of 2 regioisomers ratio ˜3/2) and PPh₃ (36.98g, 141.00 mmol, 1.4 eq) in DCM (200 mL) was added CBr₄ (46.76 g, 141.00mmol, 1.4 eq) at 0° C. and the reaction mixture was stirred at 0° C. for3 hr. TLC (petroleum ether:EtOAc=5:1) showed the starting materials wereconsumed completely, and a new spot was formed. The reaction mixture wasconcentrated in vacuo. The residue was combined with another batch (2.0g) and purified by Combiflash (from 100% of petroleum ether to 50% ofEtOAc in petroleum ether) to give desired product 22.0 g (75.53 mmol).¹H NMR (400 MHz, CHLOROFORM-d) δ ppm 7.51 (d, 1H), 6.39 (d, 1H), 5.38(s, 2H), 4.50 (s, 2H), 3.52-3.57 (m, 2H), 0.86-0.96 (m, 2H), −0.03-0.02(m, 9H).

Step D. Synthesis of4-methyl-6-((1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-3-yl)methyl)-4H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one

A suspension of4-methyl-4H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one (1.0 g,4.85 mmol, 1 eq),3-(bromomethyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazole (2.12g, 7.27 mmol, 1.5 eq), K₃PO₄ (2.57 g, 12.12 mmol, 2.5 eq) and NaI(218.05 mg, 1.45 mmol, 0.3 eq) in DMF (15 mL) was stirred at 60° C. for18 hr under N₂. TLC (petroleum ether:EtOAc=1:1) showed the startingmaterial was consumed completely, and a new spot was formed. Thereaction mixture was combined with another 3 batches (1.0 g each) andpoured into ice-water (250 mL). The mixture was extracted with EtOAc(150 mL×3). The combined organic layers were washed with water (120mL×2), brine (120 mL), and dried over Na₂SO₄. The solvent wasconcentrated in vacuo. The crude product was purified by combiflash(form 100% of petroleum ether to 80% of EtOA in petroleum ether) to givethe desired product (3.6 g). ¹H NMR (400 MHz, CHLOROFORM-d) δ ppm 8.91(s, 1H), 8.27 (s, 1H), 7.49 (d, 1H), 6.36 (d, 1H), 5.51 (s, 2H), 5.40(m, 2H), 4.45 (s, 3H), 3.52-3.58 (m, 2H), 0.85-0.90 (m, 2H), −0.05 (s,9H).

Step E. Synthesis of4-methyl-5-oxo-6-((1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-3-yl)methyl)-5,6-dihydro-4H-thiazolo[5′,4′:4,5]pyrrolo[23-d]pyridazine-2-carbaldehyde

Under argon, to a solution of 4-methyl-6-((1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-3-yl)methyl)-4H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one(1.7 g, 4.09 mmol, 1 eq) in THF (30 mL) was slowly added LiHMDS (1.0 M,8.18 mL, 2 eq) at −78° C., the reaction mixture was stirred at −70° C.for 1 hr. Then a solution of DMF (1.49 g, 20.45 mmol, 1.57 mL, 5 eq) inTHF (3 mL) was added dropwise to the mixture. The resulting mixture wasstirred at −70° C. for 1 h. TLC (petroleum ether:EtOAc=1:1) showed a newspot was formed. The reaction mixture was drop-wise added to aq. NH₄Cl(50 mL) at 0° C., then the mixture was extracted with EtOAc (30 mL×3).The combined organic layers were washed with brine (40 mL), and driedover Na₂SO₄. The solvent was removed in vacuo to afford crude desiredproduct (1.8 g) which was used for the next step without furtherpurification.

Step F. Synthesis of2-(hydroxymethyl)-4-methyl-6-((1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-3-yl)methyl)-4H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one

To a solution of4-methyl-5-oxo-6-((1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-3-yl)methyl)-5,6-dihydro-4H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazine-2-carbaldehyde(1.8 g, 3.24 mmol, 1 eq) in THF (20 mL) MeOH (10 mL) was added NaBH₄(245.08 mg, 6.48 mmol, 2 eq) at 0° C. and the reaction mixture wasstirred at room temperature for 18 hr. TLC (petroleum ether:EtOAc=1:2)showed the starting material was consumed completely, and a new spot wasformed. The reaction mixture was concentrated in vacuo, the residue waspurified by combiflash (from 100% DCM to 5% of MeOH in DCM). The desiredproduct (1.1 g) was obtained. ¹H NMR (400 MHz, DMSO-d₆) δ ppm 8.63 (s,1H), 7.86 (d, 1H), 6.44 (t, 1H), 6.27 (d, 1H), 5.40-5.42 (m, 4H), 4.98(d, 2H), 4.34 (s, 3H), 3.55-3.61 (m, 2H), 0.86-0.91 (m, 2H), 0.00 (s,9H).

Step G. Synthesis of(4-methyl-5-oxo-6-((1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-3-yl)methyl)-5,6-dihydro-4H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazin-2-yl)methylmethanesulfonate

To a solution of2-(hydroxymethyl)-4-methyl-6-((1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-3-yl)methyl)-4H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one(700 mg, 1.57 mmol, 1 eq) and Et₃N (317.21 mg, 3.13 mmol, 436.33 uL, 2.0eq) in DCM (15 mL) was added dropwise MsCl (269.32 mg, 2.35 mmol, 181.97uL, 1.5 eq) at 0° C. and the reaction mixture was stirred at roomtemperature for 1 h. TLC (petroleum ether:EtOAc=1:1) showed the startingmaterial was consumed completely, and a new spot was formed. Thereaction mixture was diluted with EtOAc (80 mL), and washed with water(30 mL×4), brine (40 mL), and dried over Na₂SO₄. The solvent was removedin vacuo to afford crude product (700 mg). LCMS: (m/z 525.5 (M+H).

Step H. Synthesis of2-((1H-1,2,4-triazol-1-yl)methyl)-4-methyl-6-((1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-3-yl)methyl)-4H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazin-5(6H)-oneand2-((4H-1,2,4-triazol-4-yl)methyl)-4-methyl-6-((1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-3-yl)methyl)-4H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one

A mixture of(4-methyl-5-oxo-6-((1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-3-yl)methyl)-5,6-dihydro-4H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazin-2-yl)methylmethanesulfonate (150 mg, 285.88 umol, 1 eq) 1H-1,2,4-triazole (197.45mg, 2.86 mmol, 10 eq) and CsF (86.85 mg, 571.77 umol, 21.08 uL, 2 eq) inMeCN (8 mL) was stirred at 60° C. under N₂ for 18 hr. LCMS showed thestarting material was consumed completely, and two new peaks wereformed. The reaction mixture was concentrated in vacuo, and the residuewas purified by Combiflash (from 100% of DCM to 8% of MeOH in DCM). Theproduct2-((1H-1,2,4-triazol-1-yl)methyl)-4-methyl-6-((1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-3-yl)methyl)-4H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one(55 mg) and2-((4H-1,2,4-triazol-4-yl)methyl)-4-methyl-6-((1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-3-yl)methyl)-4H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one(30 mg) were obtained.

Step I. Synthesis of2-((1H-1,2,4-triazol-1-yl)methyl)-6-((1H-pyrazol-3-yl)methyl)-4-methyl-4H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one

To a suspension of2-((1H-1,2,4-triazol-1-yl)methyl)-4-methyl-6-((1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-3-yl)methyl)-4H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one(55 mg, 110.52 umol, 1 eq) and HCl/dioxane (4 M, 1 mL, 36.19 eq) in DCM(3 mL) was added H₂O (1.99 mg, 110.52 umol, 0.05 mL, 1 eq) and thereaction mixture was stirred at room temperature for 18 hr. LCMS showedthe starting material was consumed completely, and 84% of desiredproduct was found. The reaction mixture was concentrated in vacuo, andthe residue was purified by Prep-HPLC to give the desired product (24.1mg, 65.60 umol, 59.35% yield). Column: Xtimate C18 150*25 mm*5 um,mobile phase: [water (0.225% FA)-ACN]; B %: 13%-43%, 11.2 min. LCMS: m/z367.9 (M+H) ¹H NMR (400 MHz, METHANOL-d₄) δ ppm 8.72 (s, 1H), 8.35 (s,1H), 8.05 (s, 1H), 7.52 (brs, 1H), 6.26 (d, 1H), 5.92 (s, 2H), 5.43 (s,2H), 4.30 (s, 3H).

Step J. Synthesis of6-((1H-pyrazol-3-yl)methyl)-2-((4H-1,2,4-triazol-4-yl)methyl)-4-methyl-4H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one

To a suspension of2-((4H-1,2,4-triazol-4-yl)methyl)-4-methyl-6-((1-((2-(trimethylsilyl)ethoxy)methyl)-H-pyrazol-3-yl)methyl)-4H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one(30 mg, 60.28 umol, 1 eq) and HCl/dioxane (4 M, 1 mL, 66.35 eq) in DCM(3 mL) was added H₂O (50.00 mg, 2.78 mmol, 0.05 mL, 46.04 eq) and thereaction mixture was stirred at room temperature for 18 hr. LCMS showedthe starting material was consumed completely, the desired product wasfound. The reaction mixture was concentrated in vacuo, and the residuewas purified by Prep-HPLC to give the desired product (2.1 mg, 5.72umol). Column: Xtimate C18 150*25 mm*5 um; mobile phase: [water (0.225%FA)-ACN];B %: 13%-43%, 11.2 min. LCMS: m/z 368.0 (M+H)⁺. ¹H NMR (400MHz, METHANOL-4) δ ppm 8.74 (s, 2H), 8.38 (s, 1H), 7.52 (d, 1H), 6.26(d, 1H), 5.84 (s, 2H), 5.44 (s, 2H), 4.31 (s, 3H).

The following compounds were synthesized according to Scheme E9 and theprocedure of Examples 9A-9B using the appropriate starting material.Standard protection and deprotection can be used when necessary.

Cpd No. Structure and chemical name Charaterization E9-13

LCMS: m/z 367.0 [M + H]⁺. ¹H NMR (400 MHz, METHANOL-d₄) δ ppm 8.39 (s,1H), 7.90 (d, 1H), 7.63 (d, 1H), 7.59 (brs, 1H), 6.43 (t, 1H), 6.29(brs, 1H), 5.85 (s, 2H), 5.48 (s, 2H), 4.37 (s, 3H). E9-14

LCMS: m/z 383.0 [M + H]⁺. ¹H NMR (400 MHz, METHANOL-d₄) δ ppm 8.41 (s,1H), 7.55-7.57 (m, 1H), 6.64 (d, 1H), 6.51 (d, 1H), 6.30 (s, 1H), 5.48(s, 2H), 5.28 (s, 2H), 4.37 (s, 3H). E9-15

LCMS: m/z 412.0 [M + H]⁺. ¹H NMR (400 MHz, DMSO-d₆) δ ppm 12.61 (brs,1H), 8.55 (s, 1H), 8.28 (d, 1H), 7.58 (brs, 1H), 7.13 (d, 1H), 6.08 (d,1H), 6.03 (s, 2H), 5.30 (brs, 2H), 4.24 (s, 3H). E9-16

LCMS: m/z 382.0 [M + H]⁺ ¹H NMR (400 MHz, METHANOL-d₄) δ ppm 8.35 (s,1H), 7.53 (d, 2H), 6.26 (brs, 1H), 5.71 (d, 1H), 5.54 (s, 2H), 5.44 (s,2H), 4.33 (s, 3H). E9-17

LCMS: m/z 366.9 [M + H]⁺. ¹HNMR (400 MHz, METHANOL-d₄) δ ppm 8.36 (s,1H), 7.89 (s, 1H), 7.54 (brs, 1H), 7.30 (s, 1H), 7.03 (s, 1H), 6.26 (s,1H), 5.72 (s, 2H), 5.44 (s, 2H), 4.33 (s, 3H). E9-18

LCMS: m/z 368 [M + H]⁺. ¹H NMR (400 MHz, DMSO-d₆) δ ppm 8.53 (brs, 1H),8.32 (s, 1H), 7.81 (s, 1H), 7.58 (brs, 1H), 6.17 (s, 2H), 6.07 (brs,1H), 5.29 (brs, 2H), 4.23 (s, 3H). E9-19

LC-MS: m/z 453 (M + H)⁺. ¹H NMR (400 MHz, DMSO-d6) δ: 10.36 (s, 1H),8.55 (s, 1H), 8.07-7.84 (m, 2H), 7.70 (t, 1H), 7.37 (s, 1H), 7.00 (s,1H), 5.79 (s, 2H), 5.45 (s, 2H), 4.26 (s, 3H), 2.01 (s, 3H)

Example 9D Synthesis of2-((1H-1,2,4-triazol-1-yl)methyl)-6-((2-aminopyrimidin-4-yl)methyl)-4-methyl-4H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one

Step A. Synthesis of2-chloro-4-methyl-4H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one

To a mixture of4-methyl-4H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one (3 g,14.5 mmol) in dry THF (80 mL) was added LiHMDS (30.5 mL,) at −65° C. bydropwise. After stirred for 1 hr, a solution of hexachloroethane (1.8mL, 16 mmol) in dry THF (20 mL) was added. The reaction mixture wasraised to −20° C. over 3 hr. Then the mixture was quenched with satd.NH₄Cl and stirred at r.t. for 20 min. The precipitate was collected byfiltration and washed with EtOAc to give 3.5 g of2-chloro-4-methyl-4H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one.LC-MS (ESI): m/z 241 (M+H)⁺.

Step B. Synthesis of4-methyl-2-vinyl-4H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one

Under nitrogen, to a mixture of2-chloro-4-methyl-4H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one(1.5 g, 6.2 mmol) and tributyl(ethenyl)stannane (5.5 mL, 18.7 mmol) inDMF (30 mL) was added Pd(PPh₃)₄(0.36 g, 0.31 mmol). The reaction mixturewas stirred at 100° C. for 2 hr. Then the mixture was cooled down anddiluted with EtOAc, washed with water and brine, dried over anhy. Na₂SO₄and concentrated. The residue was purified by flash chromatography(silica gel, 0˜10% MeOH in DCM) to give 1.4 g of4-methyl-2-vinyl-4H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one.LC-MS (ESI): m/z 233 (M+H)⁺.

Step C. Synthesis of4-methyl-5-oxo-5,6-dihydro-4H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazine-2-carbaldehyde

Under −60° C., a mixture of4-methyl-2-vinyl-4H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one(500 mg, 2.15 mmol) in DCM/MeCN (500 mL, 1:1 volume) was purged with O₃for 20 min. Then the reaction was quenched with dimethylsolfane andconcentrated to give 500 mg of4-methyl-5-oxo-5,6-dihydro-4H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazine-2-carbaldehyde.LC-MS (ESI): m/z 235 (M+H)⁺.

Step D. Synthesis of2-(hydroxymethyl)-4-methyl-4H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one

To a mixture of4-methyl-5-oxo-5,6-dihydro-4H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazine-2-carbaldehyde(500 mg, 2.13 mmol) in EtOH (3 mL) was added NaBH₄ (81 mg, 2.13 mmol) at0° C. The reaction was stirred at room temperature for 5 min. Then themixture was poured into satd. NH₄Cl, extracted with EtOAc. The organiclayer was washed with brine, dried over anhy. Na₂SO₄ and concentrated.The residue was purified by flash chromatography (silica gel, 0˜8% MeOHin DCM) to give 120 mg of2-(hydroxymethyl)-4-methyl-4H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one.LC-MS (ESI): m/z 237 (M+H)⁺.

Step E. Synthesis of6-((2-chloropyrimidin-4-yl)methyl)-2-(hydroxymethyl)-4-methyl-4H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one

To a mixture of2-(hydroxymethyl)-4-methyl-4H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one(200 mg, 0.85 mmol) in DMF (8 mL) was added K₂CO₃ (351 mg, 2.54 mmol).After stirred at 60° C. for 30 min, a solution of2-chloro-4-(chloromethyl)pyrimidine (276 mg, 1.7 mmol) in DMF (2 mL) wasadded. The reaction mixture was stirred for another 4 hr, poured intosatd. NH₄Cl, extracted with EtOAc. The organic layer was washed withbrine, dried over anhy. Na₂SO₄ and concentrated. The residue waspurified by flash chromatography (silica gel, 0˜8% MeOH in DCM) to give160 mg of6-((2-chloropyrimidin-4-yl)methyl)-2-(hydroxymethyl)-4-methyl-4H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one.LC-MS (ESI): m/z 363 (M+H)⁺.

Step F. Synthesis of6-((2-(bis(2,4-dimethoxybenzyl)amino)pyrimidin-4-yl)methyl)-2-(hydroxymethyl)-4-methyl-4H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one

To a mixture of6-((2-chloropyrimidin-4-yl)methyl)-2-(hydroxymethyl)-4-methyl-4H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one(160 mg, 0.44 mmol) in MeCN (5 mL) was addedbis(2,4-dimethoxybenzyl)amine (280 mg, 0.88 mmol) and AcOH (1 drop). Thereaction mixture was stirred at 80° C. overnight. The reaction mixturewas evaporated and the residue was purified by prep-TLC (eluant: 5% MeOHin DCM) to give 75 mg of6-((2-(bis(2,4-dimethoxybenzyl)amino)pyrimidin-4-yl)methyl)-2-(hydroxymethyl)-4-methyl-4H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one.LC-MS (ESI): m/z 644 (M+H)⁺.

Step G. Synthesis of6-((2-(bis(2,4-dimethoxybenzyl)amino)pyrimidin-4-yl)methyl)-2-(chloromethyl)-4-methyl-4H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one

Under 0° C., to a solution of6-((2-(bis(2,4-dimethoxybenzyl)amino)pyrimidin-4-yl)methyl)-2-(hydroxymethyl)-4-methyl-4H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one(75 mg, 0.12 mmol) in DCM (5 mL) was added Et₃N (0.16 mL, 1.16 mmol) andMsCl (0.05 mL, 0.58 mmol). The mixture was stirred at r.t. overnight.The reaction mixture was diluted with DCM, washed with satd. NH₄Cl andbrine, dried over anhy. Na₂SO₄ and concentrated to give 70 mg of crudeproduct of6-((2-(bis(2,4-dimethoxybenzyl)amino)pyrimidin-4-yl)methyl)-2-(chloromethyl)-4-methyl-4H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one.LC-MS (ESI): m/z 662 (M+H)⁺.

Step H. Synthesis of2-((1H-1,2,4-triazol-1-yl)methyl)-6-((2-(bis(2,4-dimethoxybenzyl)amino)pyrimidin-4-yl)methyl)-4-methyl-4H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one

A mixture of 4H-1,2,4-triazole (39 mg, 0.57 mmol) and K₂CO₃ (78 mg, 0.57mmol) in DMF (3 mL) was stirred at 60° C. for 30 min.6-((2-(bis(2,4-dimethoxybenzyl)amino)pyrimidin-4-yl)methyl)-2-(chloromethyl)-4-methyl-4H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one(75 mg, 0.11 mmol) was added and stirred for another 30 min. Thesuspension was poured into satd. NH₄Cl, extracted with EtOAc. Theorganic layer was washed with brine, dried over anhy. Na₂SO₄ andconcentrated. The residue was purified by flash chromatography (silicagel, 0˜10% DCM in MeOH) to give 60 mg of2-((1H-1,2,4-triazol-1-yl)methyl)-6-((2-(bis(2,4-dimethoxybenzyl)amino)pyrimidin-4-yl)methyl)-4-methyl-4H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one.LC-MS (ESI): m/z 695 (M+H)⁺.

Step G. Synthesis of2-((1H-1,2,4-triazol-1-yl)methyl)-6-((2-aminopyrimidin-4-yl)methyl)-4-methyl-4H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one

To a mixture of 2-((1H-1,2,4-triazol-1-yl)methyl)-6-((2-(bis(2,4-dimethoxybenzyl)amino)pyrimidin-4-yl)methyl)-4-methyl-4H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazin-5(6H)-One(30 mg, 0.043 mmol) in EtOH (2 mL) was added HCl (0.5 mL, 4 M indioxane). The reaction mixture was stirred at 80° C. overnight. Then themixture was cooled down and poured into satd. NaHCO₃, extracted withEtOAc. The organic layer was washed with brine, dried over anhy. Na₂SO₄and concentrated. The residue was purified by prep-HPLC to give 8 mg of2-((1H-1,2,4-triazol-1-yl)methyl)-6-((2-aminopyrimidin-4-yl)methyl)-4-methyl-4H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one.LC-MS (ESI): m/z 395 (M+H)⁺. ¹H NMR (400 MHz, DMSO-d₆) δ 8.80 (s, 1H),8.62 (s, 1H), 8.12-8.10 (m, 2H), 6.60 (s, 2H), 6.19 (d, 1H), 6.01 (s,2H), 5.19 (s, 2H), 4.26 (s, 3H).

Cpd No. Structure and chemical name Charaterization E9-31

LC-MS: m/z 394 (M + H)⁺. ¹H NMR (400 MHz, DMSO-d6) δ: 8.62 (s, 1H), 8.11(d, 1H), 7.88 (s, 1H), 7.36 (s, 1H), 6.99 (s, 1H), 6.61 (s, 2H), 6.18(d, 1H), 5.79 (s, 2H), 5.19 (s, 2H), 4.26 (s, 3H). E9-32

LC-MS: m/z 385 (M + H)⁺. ¹H NMR (400 MHz, DMSO-d6) δ: 11.32-11.28 (m,2H), 8.80 (s, 1H), 8.59 (s, 1H), 8.11 (s, 1H), 6.01 (s, 2H), 5.14 (s,2H), 4.26 (s, 3H).

Example 10. Synthesis of Compounds E10-ii

Reaction of E10-i with LiHMDS and an aldehyde generates compound E10-ii,which can be separated with chiral HPLC or SFC to give two enantiomers.As used herein, Ar_(t) and Ar₂ are each independently optionallysubstituted 5- or 6-membered monocyclic heteroaryl.

Example 10A. Synthesis of(R)-6-((1H-pyrazol-3-yl)methyl)-2-(hydroxy(1H-pyrazol-3-yl)methyl)-4-methyl-4H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazin-5(6H)-oneand(S)-6-((1H-pyrazol-3-yl)methyl)-2-(hydroxy(1H-pyrazol-3-yl)methyl)-4-methyl-4H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one

Step A. Synthesis of2-(hydroxy(1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-3-yl)methyl)-4-methyl-6-((1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-3-yl)methyl)-4H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one

Under argon, to a solution of4-methyl-6-((1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-3-yl)methyl)-4H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one(0.5 g, 1.20 mmol, 1 eq) in THF (10 mL) was slowly added LiHMDS (1.0 M,2.41 mL, 2 eq) at −78° C., and the reaction mixture was stirred at −70°C. for 1 hr. Then a solution of 1-(2-trimethylsilylethoxymethyl)pyrazole-3-carbaldehyde (816.97 mg, 3.61 mmol, 3 eq) in THF (1 mL) wasadded to the reaction mixture. The resulting mixture was stirred at −70°C. for 1 hr. TLC (petroleumether:EtOAc=1:1) showed two new spot wasformed. The reaction mixture was quenched by aq NH₄Cl (5 mL) at −70° C.,and then warmed to room temperature. The mixture was diluted with water(10 mL), and extracted with EtOAc (8 mL×3). The combined organic layerswere washed with brine (10 mL), and dried over Na₂SO₄. The solvent wasconcentrated in vacuo. The residue was purified by Combiflash (from 100%of petroleum ether to 100% of EtOAc) to give crude product (130 mg) aspale brown gum, which was used for the next step without furtherpurification. LCMS: m/z 643.2 [M+H]

Step B. Synthesis of6-((1H-pyrazol-3-yl)methyl)-2-(hydroxy(1H-pyrazol-3-yl)methyl)-4-methyl-4H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one

To a solution of2-(hydroxy(1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-3-yl)methyl)-4-methyl-6-((1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazol-3-yl)methyl)-4H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one(0.13 g, 202.20 umol, 1 eq) in DCM (8 mL) was added TFA (4.62 g, 40.52mmol, 3 mL, 200.38 eq), followed by H₂O (500.00 mg, 27.75 mmol, 0.5 mL,137.26 eq), then the reaction mixture was stirred at room temperaturefor 18 hr, then heated to 40° C. for 18 hr. LCMS showed the startingmaterial was consumed completely. The reaction mixture was concentratedin vacuo, and the residue was purified by Prep-HPLC to give the desiredproduct (20.5 mg). LCMS: m/z 382.9 [M+H]. ¹H NMR (400 MHz, DMSO-d₆) δppm 12.72 (brs, 1H), 12.61 (brs, 1H), 8.52 (s, 1H), 7.56-7.62 (m, 2H),6.82 (brs, 1H), 6.18 (d, 1H), 5.99-6.09 (m, 2H), 5.24-5.32 (m, 2H), 4.19(s, 3H).

Step C. Synthesis of(R)-6-((1H-pyrazol-3-yl)methyl)-2-(hydroxy(1H-pyrazol-3-yl)methyl)-4-methyl-4H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazin-5(6H)-oneand(S)-6-((1H-pyrazol-3-yl)methyl)-2-(hydroxy(1H-pyrazol-3-yl)methyl)-4-methyl-4H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazin-5(6H)-one

The compound6-((1H-pyrazol-3-yl)methyl)-2-(hydroxy(1H-pyrazol-3-yl)methyl)-4-methyl-4H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazin-5(6H)-onewas separated by SFC. SFC condition: Column is DAICEL CHIRALCEL OJ-H(250 mm*30 mm, Sum); mobile phase: A: 55% of CO2; B: 45% [0.1% NH₃H₂O inEtOH]/min. The SFC separation afforded2-[(R)-hydroxy(1H-pyrazol-3-yl)methyl]-4-methyl-6-(1H-pyrazol-3-ylmethyl)thiazolo[3,4]pyrrolo[1,3-d]pyridazin-5-oneand2-[(S)-hydroxy(1H-pyrazol-3-yl)methyl]-4-methyl-6-(1H-pyrazol-3-ylmethyl)thiazolo[3,4]pyrrolo[1,3-d]pyridazin-5-one.One isomer (4.4 mg): LCMS: m/z 383 [M+H]⁺. ¹H NMR (400 MHz, Methanol-d₄)δ ppm 8.39 (s, 1H), 7.61 (brs, 1H), 7.55 (brs, 1H), 6.34 (brs, 1H), 6.25(brs, 1H), 6.18 (brs, 1H), 5.44 (s, 2H), 4.29 (s, 3H). Another isomer(4.1 mg): LCMS: m/z 383 (M+H)⁺. ¹H NMR (400 MHz, METHANOL-d₄) δ ppm 8.40(s, 1H), 7.59 (brs, 1H), 7.54 (brs, 1H), 6.34 (brs, 1H), 6.26 (brs, 1H),6.19 (brs, 1H), 5.44 (s, 2H), 4.29 (s, 3H).

Example 10B: Synthesis of6-((1H-pyrazol-3-yl)methyl)-2-(difluoro(1H-pyrazol-3-yl)methyl)-4-methyl-4,6-dihydro-5H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazin-5-one

Step A.3-(6-((1-(N,N-dimethylsulfamoyl)-1H-pyrazol-3-yl)methyl)-4-methyl-5-oxo-5,6-dihydro-4H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazine-2-carbonyl)-N,N-dimethyl-1H-pyrazole-1-sulfonamide

To a mixture of3-((2-((1-(N,N-dimethylsulfamoyl)-1H-pyrazol-3-yl)(hydroxy)methyl)-4-methyl-5-oxo-4,5-dihydro-6H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazin-6-yl)methyl)-N,N-dimethyl-1H-pyrazole-1-sulfonamide(50 mg, 83.80 umol, made similarly to E10-1) in DCM (1.5 mL) was addedMnO₂ (72.85 mg, 838.00 umol) and the mixture was stirred at 15° C. for1.5 hours. The reaction mixture was filtered and concentrated underreduced pressure to give a crude product (60 mg, crude). LCMS: m/z 595.1(M+H)⁺. ¹H NMR (400 MHz, CDCl3) δ 8.34 (s, 1H), 8.12 (d, 1H), 7.90 (d,1H), 7.41 (d, 1H), 6.38 (d, 1H), 5.54 (s, 2H), 4.49 (s, 3H), 3.10 (s,6H) 2.93 (s, 6H).

Step B.3-((2-((1-(N,N-dimethylsulfamoyl)-1H-pyrazol-3-yl)difluoromethyl)-4-methyl-5-oxo-4,5-dihydro-6H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazin-6-yl)methyl)-N,N-dimethyl-1H-pyrazole-1-sulfonamide

To a solution of3-(6-((1-(N,N-dimethylsulfamoyl)-1H-pyrazol-3-yl)methyl)-4-methyl-5-oxo-5,6-dihydro-4H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazine-2-carbonyl)-N,N-dimethyl-1H-pyrazole-1-sulfonamide(240 mg, 403.60 umol) in DCE (4 mL) was added BAST (1.34 g, 6.05 mmol,1.33 mL), and the mixture was stirred at 50° C. for 12 h. The reactionmixture was diluted with dichloromethane (20 mL) and washed withsaturated NaHCO₃ (10 mL*2), dried over anhydrous Na₂SO₄, filtered andconcentrated under reduced pressure to give a crude product (300 mg,crude) which was used in the next step without further purification.LCMS: m/z 617.1 (M+H)⁺.

Step C.6-((1H-pyrazol-3-yl)methyl)-2-(difluoro(1H-pyrazol-3-yl)methyl)-4-methyl-4,6-dihydro-5H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazin-5-one

To a mixture of3-((2-((1-(N,N-dimethylsulfamoyl)-1H-pyrazol-3-yl)difluoromethyl)-4-methyl-5-oxo-4,5-dihydro-6H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazin-6-yl)methyl)-N,N-dimethyl-1H-pyrazole-1-sulfonamide(240 mg, 163.47 umol) in DCM (2 mL) was added TFA (2.07 g, 18.15 mmol,1.34 mL) and the mixture was warmed up to 50° C. for 5 h. The reactionmixture was concentrated under reduced pressure. The residue waspurified by prep-HPLC (column: Agela ASB 150*25 mm*5 um; mobile phase:[water (0.05% HCl)-ACN]; B %: 30%-60%, 8 min) to give desired product(3.9 mg, 5.45% yield, 92% purity) as a white solid. LCMS: m/z 403.1(M+H)⁺. ¹H NMR (400 MHz, DMSO-d₆) δ 8.67 (s, 1H), 7.95 (s, 1H), 7.61 (s,1H), 6.72 (s, 1H), 6.16 (s, 1H), 5.36 (s, 2H), 4.28 (s, 3H).

Example 10C: Synthesis of2-(1-(1H-pyrazol-3-yl)ethyl)-6-((1H-pyrazol-3-yl)methyl)-4-methyl-4,6-dihydro-5H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazin-5-one

Step A:3-((2-(1-(1-(N,N-dimethylsulfamoyl)-1H-pyrazol-3-yl)-1-hydroxyethyl)-4-methyl-5-oxo-4,5-dihydro-6H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazin-6-yl)methyl)-N,N-dimethyl-1H-pyrazole-1-sulfonamide

To a solution of3-(6-((1-(N,N-dimethylsulfamoyl)-1H-pyrazol-3-yl)methyl)-4-methyl-5-oxo-5,6-dihydro-4H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazine-2-carbonyl)-N,N-dimethyl-1H-pyrazole-1-sulfonamide(160 mg, 269.07 umol) in THF (3 mL) was added CH3MgBr (3 M, 179.38 uL)and the reaction mixture was stirred at 0° C. for 3 h. The reactionmixture was poured into saturated NH4Cl (10 mL) at 0° C., extracted withethyl acetate (20 mL*3). The combined organic layers were washed withbrine (10 mL), dried over anhydrous Na2SO4, filtered and concentratedunder reduced pressure. The residue was purified by flash silica gelchromatography (ISCO®; 12 g SepaFlash® Silica Flash Column, Eluent of0-90% Ethyl acetate/Petroleum ether gradient @ 40 mL/min) to givedesired product (50 mg, 81.87 umol). LCMS: m/z 611.1 (M+H)⁺.

Step B:2-(1-(1H-pyrazol-3-yl)ethyl)-6-((1H-pyrazol-3-yl)methyl)-4-methyl-4,6-dihydro-5H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazin-5-one

To a mixture of3-((2-(1-(1-(N,N-dimethylsulfamoyl)-1H-pyrazol-3-yl)-1-hydroxyethyl)-4-methyl-5-oxo-4,5-dihydro-6H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazin-6-yl)methyl)-N,N-dimethyl-1H-pyrazole-1-sulfonamide(50 mg, 81.87 umol) in DCE (0.5 mL) was added Et3SiH (19.04 mg, 163.75umol, 26.15 uL) at 0° C., followed by TFA (1.54 g, 13.51 mmol, 1 mL,164.96 eq) and stirred at 0° C. for 1 h. The mixture was warmed up to50° C. for another 1 h. The reaction mixture was concentrated underreduced pressure. The residue was purified by prep-HPLC (column: AgelaASB 150*25 mm*5 um; mobile phase: [water (0.05% HCl)-ACN];B %: 25%-55%,7 min) to give 7.0 mg of desired product. LCMS: m/z 381.2 (M+H)⁺. ¹H NMR(400 MHz, CD₃OD) δ 8.46 (s, 1H), 8.15 (d, 2H), 6.78 (d, 1H), 6.74 (d,1H), 5.60 (s, 2H), 5.00 (q, 1H), 4.33 (s, 3H), 1.94 (d, 3H).

Example 10D: Synthesis of6-((1H-pyrazol-3-yl)methyl)-4-methyl-2-(1H-pyrazole-3-carbonyl)-4,6-dihydro-5H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazin-5-one

Step A.4-methyl-2-(1H-pyrazole-3-carbonyl)-6-(1H-pyrazol-3-ylmethyl)thiazolo[3,4]pyrrolo[1,3-d]pyridazin-5-one

To a solution of3-(6-((1-(N,N-dimethylsulfamoyl)-1H-pyrazol-3-yl)methyl)-4-methyl-5-oxo-5,6-dihydro-4H-thiazolo[5′,4′:4,5]pyrrolo[2,3-d]pyridazine-2-carbonyl)-N,N-dimethyl-1H-pyrazole-1-sulfonamide(50 mg, 84.08 umol) in DCE (1.5 mL) was added TFA (2.31 g, 20.26 mmol,1.5 mL) and the reaction mixture was warmed up to 50° C. for 12 h. Thereaction mixture was concentrated under vacuum. The residue was purifiedby prep-HPLC to give 6.0 mg of desired product. LCMS: m/z 381.1 (M+H)⁺.¹H NMR (400 MHz, DMSO-d₆) δ 8.72 (s, 1H), 7.96 (d, 1H), 7.64 (d, 1H),7.49 (d, 1H), 6.19 (d, 1H), 5.37 (s, 2H), 4.38 (s, 3H).

Example 11. PKR Mutant Assay Procedure:

PKR or PKR mutant enzyme solution was diluted into a reaction mix, whichcontains 1× buffer (100 mM KCl, 50 mM Tris pH 7.5, 5 mM MgCl₂) as wellas PEP (conc depends on enzyme), 180 μM NADH, 0.5 units LDH, 1 mM DTT,0.03% BSA; final assay concentrations after 1.11 fold dilution areindicated.

2 μL of test compound was added into wells first, and then 180 μLreaction mix was added.

Reactions mixture with test compound was assembled except for ADP, andplates were stored for 60 minutes at room temperature.

20 uL ADP was added to start reaction at room temperature and reactionprogress was measured as changes in absorbance at 340 nm wavelength atroom temperature.

Test Compound Preparation:

Test compound stock was made at 100× concentration in 100% DMSO (10 mM)

1 to 3 dilutions were made for 11 points (i.e. 50p of firstconcentration added to 100 μl 100% DMSO to yield 3.33 mM, 50 μl of thisadded to 100 μl DMSO to yield 1.11 mM, and so forth)

-   -   1 to 100 dilution into assay (2 μl in 200 μl) yielded starting        concentration of 100 μM, decreasing 3 fold for 11 points.

Assay Buffer: 100 mM KCl, 50 mM Tris 7.5, 5 mM MgCl2, 1 mM DTT, 0.03%BSA

Reaction Mixture: PKR mutant enzyme: 40-400 ng/well; ADP: 0.2-1.65 mM;PEP: 0.1-0.5 mM; NADH: 180 μM; LDH: 0.5 units (Sigma #59023); DTT: 1 mM;BSA: 0.03%.

Example 12. PKR WT Single Point Percent Activation Assay

A compound described herein was diluted with DMSO and tested at 1 μMconcentration. The enzyme was diluted in an enzyme solution, whichcontains 1× buffer (100 mM KCl, 50 mM Tris pH 7.5, 5 mM MgCl₂) as wellas PEP (conc depends on enzyme), 180 μM NADH, 0.5 units LDH, 1 mM DTT,0.03% BSA; final assay concentrations after 1.11 fold dilution areindicated.

2 μL of compound solution was first added into wells, and then 180 μL ofenzyme solution was added. Assays were assembled except for ADP, andplates were stored for 60 minutes at RT. 20 μL ADP was added to startthe assay and assay output was evaluated using OD340. The assay was runat room temperature.

Final concentration: PKR wt (100 ng/well), Tris pH 7.5 (50 mM), KCl (100mM), MgCl₂ (5 mM), ADP (0.48 mM), PEP (0.15 mM), NADH (180 μM), LDH (0.5units, Sigma 59023), DTT (1 mM) and BSA (0.03%).

Example 13. PKR R510Q Single Point Percent Activation Assay

A compound described herein was diluted with DMSO and tested at 1 μMconcentration. The enzyme was diluted in into an enzyme solution, whichcontains 1× buffer (100 mM KCl, 50 mM Tris pH 7.5, 5 mM MgCl2) as wellas PEP (conc depends on enzyme), 180 μM NADH, 0.5 units LDH, 1 mM DTT,0.03% BSA; final assay concentrations after 1.11 fold dilution areindicated.

2 μL of compound solution was first added into wells, and then 180 μL ofenzyme. solution was added. Assays were assembled except for ADP, andplates were stored for 60 minutes at RT. 20 μL ADP was added to startthe assay and assay output was evaluated using OD340. The assay was runat room temperature.

Final concentration: PKR R510Q (40 ng/well), Tris pH 7.5 (50 mM), KCl(100 mM), MgCl₂ (5 mM), ADP (0.2 mM), PEP (0.11 mM), NADH (180 μM), LDH(0.5 units, Sigma 59023), DTT (1 mM) and BSA (0.03%).

Example 14. PKR R532W Single Point Percent Activation Assay

A compound described herein was diluted with DMSO and tested at 1 μMconcentration. The enzyme was diluted in an enzyme solution, whichcontains 1× buffer (100 mM KCl, 50 mM Tris pH 7.5, 5 mM MgCl2) as wellas PEP (conc depends on enzyme), 180 μM NADH, 0.5 units LDH, 1 mM DTT,0.03% BSA; final assay concentrations after 1.11 fold dilution areindicated.

2 μL of compound solution was first added into wells, and then 180 μL ofenzyme solution was added. Assays were assembled except for ADP, andplates were stored for 60 minutes at RT. 20 μL ADP was added to startthe assay and assay output was evaluated using OD340. The assay was runat room temperature.

Final concentration: PKR R532W (100 ng/well), Tris pH 7.5 (50 mM), KCl(100 mM), MgCl2 (5 mM), ADP (0.36 mM), PEP (0.1 mM), NADH (180 μM), LDH(0.5 units, Sigma 59023), DTT (1 mM) and BSA (0.03%).

Example 15. PKR T384M Single Point Percent Activation Assay

A compound described herein was diluted with DMSO and tested at 1 μMconcentration. The enzyme was diluted in into an enzyme solution, whichcontains 1× buffer (100 mM KCl, 50 mM Tris pH 7.5, 5 mM MgCl2) as wellas PEP (conc depends on enzyme), 180 μM NADH, 0.5 units LDH, 1 mM DTT,0.03% BSA; final assay concentrations after 1.11 fold dilution areindicated.

2 μL of compound solution was first added into wells, and then 180 μLenzyme solution was added. Assays were assembled except for ADP, andplates were stored for 60 minutes at RT. 20 μL ADP was added to startthe assay and assay output was evaluated using OD340. The assay was runat room temperature.

Final concentration: PKR T384M soluble (300 ng/well), Tris pH 7.5 (50mM), KCl (100 mM), MgCl2 (5 mM), ADP (0.08 mM), PEP (0.23 mM), NADH (180μM), LDH (0.5 units, Sigma 59023), DTT (1 mM) and BSA (0.03%).

Example 16. Red Blood Cell (RBC) Purification

Fresh blood drawn from healthy volunteers into K₂EDTA tubes wascollected. Whole blood was pelleted by spinning at 500 g for 10 minutes.Cut transfusion bag port off of Purecell leukocyte reduction neofilter(Fisher NC0267633) one (1) inch above filter. Attach a 10 ml syringebarrel to the remaining cut tubing attached to neofilter. The plasmalayer was removed from the pellet of the whole blood and the pellet wasresuspended in 2× volume of phosphate buffered saline (PBS). Transfer 9ml re-suspended blood cell pellet to prepared 10 ml syringe that isattached to the neofilter. Allow whole blood to gravity flow throughfilter until all fluid runs through upper tubing into filter disc. Addplunger to the syringe, remove syringe from clamp and invert the filter,then plunge air through the syringe filter system. Using a new 5 mlsyringe, remove filtered RBCs from the bag by the syringe port andtransfer purified RBCs to a 5 ml snap cap tube that has been incubatedon ice. Spin 5 ml snap cap tube at 500 g for 10 minutes at 15C, aspiratesupernatant and resuspend in AGAM (1×PBS, 1% glucose, 170 mg/L adenine,5.25 g/L mannitol) at a density of 4×10⁹ cells/mL.

Example 17. Cell Based ATP Assay

For cell based ATP assays, the compound as described herein was preparedin 100% DMSO as a 10 mM stock. Serial dilutions (1:4) were performed in96-well V-bottom storage plate and then added 1:100 to 96-well V-bottomplates containing AGAM. RBCs were diluted in AGAM media to a density of1×10⁷ cells/mL before added 90 μL/well to black clear bottom assayplates (final compound concentration at 0.1% DMSO concentration). Assayplates were sealed using aluminum foil seals and incubated overnight at37° C. in a humidified chamber. ATP levels were read out usingCell-Titer-Glo (Promega).

Having thus described several aspects of several embodiments, it is tobe appreciated various alterations, modifications, and improvements willreadily occur to those skilled in the art. Such alterations,modifications, and improvements are intended to be part of thisdisclosure, and are intended to be within the spirit and scope of theinvention. Accordingly, the foregoing description and drawings are byway of example only.

1. A compound represented by the following structural formula:

or a pharmaceutically acceptable salt thereof, wherein: R¹ is hydrogen,an optionally substituted alkyl, an optionally substituted haloalkyl, anoptionally substituted alkenyl, an optionally substituted alkynyl, anoptionally substituted cycloalkyl, an optionally substitutedheterocyclyl, an optionally substituted aryl, —OR^(o1), —C(═O)R^(c1), ora nitrogen protecting group; wherein: R^(o1) is hydrogen, optionallysubstituted alkyl, or an oxygen protecting group; R^(c1) is optionallysubstituted alkyl or —N(R^(cn))₂, wherein each instance of R^(cn) isindependently hydrogen, —C₁₋₆ alkyl, or a nitrogen protecting group; R²and Q are each independently an optionally substituted 5- or 6-memberedmonocyclic heteroaryl; R^(a) and R^(b) are each independently hydrogen,a halogen, —CN, —NO₂, —N₃, an optionally substituted alkyl, —OR^(o3),—N(R^(n1))₂, —C(═O)N(R^(n1))₂, or C(═O)R^(c2); or alternatively R^(a)and R^(b) can be taken together with the carbon atom to which they areattached to form an optionally substituted cycloalkyl or an optionallysubstituted heterocyclyl; wherein: each instance of R^(n1) isindependently hydrogen, an optionally substituted —C₁-C₆ alkyl, or anitrogen protecting group; R^(o3) is hydrogen, an optionally substituted—C₁-C₆ alkyl, or an oxygen protecting group; and R^(c2) is an optionallysubstituted —C₁-C₆ alkyl; and R^(j) and R^(k) are each independentlyhydrogen, a halogen, —CN, —OR^(o7), —N(R^(n5))₂, —N(R^(n5))C(═O) R^(c5),—C(═O)N(R^(n5))₂, —C(═O)R^(c5), —C(═O)OR^(o7), SR^(js), —S(═O)₂R^(js),—S(═O)R^(js), or an optionally substituted —C₁-C₆ alkyl; oralternatively R^(j) and R^(k) can be taken together with the carbon atomto which they are attached to form C═O, an optionally substituted C₁-C₆monocyclic cycloalkyl ring, or an optionally substituted C₃-C₆monocyclic heterocyclyl ring; wherein: each instance of R^(n5) isindependently hydrogen, an optionally substituted —C₁-C₆ alkyl,—OR^(o8), or a nitrogen protecting group, wherein R^(o8) is hydrogen, anoptionally substituted —C₁-C₆ alkyl, or an oxygen protecting group; eachinstance of R^(o7) is independently hydrogen, an optionally substituted—C₁-C₆ alkyl, or an oxygen protecting group; each instance of R^(c5) isindependently an optionally substituted —C₁-C₆ alkyl; and each instanceof R^(js) is independently an optionally substituted —C₁-C₆ alkyl, anoptionally substituted C₆₋₁₂ aryl, an optionally substituted heteroaryl,or a sulfur protecting group.
 2. The compound according to claim 1 or apharmaceutically acceptable salt thereof, wherein the 5- or 6-memberedmonocyclic heteroaryl represented by R² is optionally substituted ateach substitutable ring carbon atom by R^(p) and optionally substitutedat each substitutable ring nitrogen atom by R^(n6); wherein: eachinstance of R^(p) is independently hydrogen, a halogen, —CN, —NO₂, —N₃,an optionally substituted alkyl, an optionally substituted alkenyl, anoptionally substituted alkynyl, an optionally substituted cycloalkyl, anoptionally substituted aryl, an optionally substituted heterocyclyl, anoptionally substituted heteroaryl, —OR^(o6), —SR^(s2), —N(R^(n3))₂,—C(═O)N(R^(n3))₂, —N(R^(n3))C(═O)R^(c4), —C(═O)R^(c4), —C(═O)OR^(o6),—OC(═O)R^(c4), —S(═O)R^(s2), —S(═O)₂R^(s2), —S(═O)OR^(o6),—OS(═O)R^(c4), —S(═O)₂OR^(o6), —OS(═O)₂R^(c4), —S(═O)N(R^(n3))₂,—S(═O)₂N(R^(n3))₂, —N(R^(n3))S(═O)R^(s2), —N(R^(n3))S(═O)₂R^(s2),—N(R^(n3))C(═O)OR^(o6), —OC(═O)N(R^(n3))₂, —N(R^(n3))C(═O)N(R^(n3))₂,—N(R^(n3))S(═O)N(R^(n3))₂, —N(R^(n3))S(═O)₂N(R^(n3))₂,—N(R^(n3))S(═O)OR^(o6), —N(R^(n3))S(═O)₂OR^(o6), —OS(═O)N(R^(n3))₂, or—OS(═O)₂N(R^(n3))₂; or alternatively two instances of R^(p) attached tothe same or adjacent carbon atoms, can be taken together with the carbonatom(s) to which they are attached to form an optionally substitutedcycloalkyl or a heterocycloalkyl; wherein: each instance of R^(n3) isindependently hydrogen, an optionally substituted —C₁-C₆ alkyl, or anitrogen protecting group; each instance of R^(o6) is independentlyhydrogen, an optionally substituted —C₁-C₆ alkyl, or an oxygenprotecting group; and each instance of R⁴ is an optionally substituted—C₁-C₆ alkyl; each instance of R^(s2) is independently an optionallysubstituted —C₁-C₆ alkyl or a sulfur protecting group; and R^(n6) ishydrogen, an optionally substituted —C₁₋₆ alkyl, or a nitrogenprotecting group.
 3. The compound according to any one of claims 1 and 2or a pharmaceutically acceptable salt thereof, wherein the 5- or6-membered monocyclic heteroaryl represented by R² is selected from oneof the following:

wherein: each instance of R^(nc) and R^(nd) is independently hydrogen,optionally substituted —C₁-C₆ alkyl, or a nitrogen protecting group; andp is 0, 1, 2, 3, or 4, as valency permits.
 4. The compound according toany one of claims 1-3 or a pharmaceutically acceptable salt thereof,wherein the 5- or 6-membered monocyclic heteroaryl represented by R² isselected from one of the following:


5. The compound according to any one of claims 1-4 or a pharmaceuticallyacceptable salt thereof, wherein the 5- or 6-membered monocyclicheteroaryl represented by R² is selected from one of the following:


6. The compound according to any one of claims 1-5, wherein the compoundis represented by the following structural formula:

or a pharmaceutically acceptable salt thereof, wherein q is 0, 1, 2, or3.
 7. The compound according to any one of claims 1-5, wherein thecompound is represented by the following structural formula:

or a pharmaceutically acceptable salt thereof, wherein q is 0, 1, 2, or3.
 8. The compound according to any one of claims 1-5, wherein thecompound is represented by the following structural formula:

or a pharmaceutically acceptable salt thereof, wherein q is 0, 1, 2, or3.
 9. The compound according to any one of claims 1-5, wherein thecompound is represented by the following structural formula:

or a pharmaceutically acceptable salt thereof, wherein q is 0, 1, 2, or3.
 10. The compound according to any one of claims 2-9 or apharmaceutically acceptable salt thereof, wherein R^(n6) is hydrogen ora —C₁₋₄ alkyl.
 11. The compound according to any one of claims 2-7 or apharmaceutically acceptable salt thereof, wherein each instance of R^(p)is independently hydrogen, halogen, optionally substituted C₁₋₄ alkyl,—CN, —NO₂, —N₃, —OR^(o4), —N(R^(n2))₂, —C(═O)N(R^(n2))₂, —C(═O)R^(c3),or —C(═O)OR^(o4).
 12. The compound according to any one of claims 1-11or a pharmaceutically acceptable salt thereof, wherein the 5- or6-membered monocyclic heteroaryl represented by Q is selected from thefollowing:

wherein: each instance of R^(n) is independently hydrogen, a halogen,—CN, —NO₂, —N₃, an optionally substituted alkyl, an optionallysubstituted alkenyl, an optionally substituted alkynyl, an optionallysubstituted cycloalkyl, an optionally substituted aryl, an optionallysubstituted heterocyclyl, an optionally substituted heteroaryl,—OR^(o4), —SR^(s1), —N(R^(n2))₂, —C(═O)N(R^(n2))²,—N(R^(n2))C(═O)R^(c3), —C(═O)R^(c3), —C(═O)OR^(o4), —OC(═O)R^(c3),—S(═O)R^(s1), —S(═O)₂R^(s1), —S(═O)OR^(o4), —OS(═O)R^(c3),—S(═O)₂OR^(o4), —OS(═O)₂R^(c3), —S(═O)N(R^(n2))₂, —S(═O)₂N(R^(n2))₂,—N(R^(n2))S(═O)R^(s1), —N(R^(n2))S(═O)₂R^(s1), —N(R^(n2))C(═O)OR^(o4),—OC(═O)N(R^(n2))₂, —N(R^(n2))C(═O)N(R^(n2))₂, —N(R^(n2))S(═O)N(R^(n2))₂,—N(R^(n2))S(═O)S(═O)₂N(R^(n2))₂, —N(R^(n2))S(O)OR^(o4),—N(R^(n2))S(═O)₂OR^(o4), —OS(═O)N(R^(n2))₂, or —OS(═O)₂N(R^(n2))₂; ortwo each instance of R^(n) attached to the same or adjacent carbonatoms, taken together with the carbon atoms to which they are attachedto form an optionally substituted cycloalkyl or a heterocycloalkyl;wherein: each instance of R^(n2) is independently hydrogen, anoptionally substituted —C₁-C₆ alkyl, or a nitrogen protecting group;each instance of R^(o4) is independently hydrogen, an optionallysubstituted —C₁-C₆ alkyl, or an oxygen protecting group; each instanceof R^(c3) is independently an optionally substituted —C₁-C₆ alkyl; eachinstance of R^(s1) is independently an optionally substituted —C₁-C₆alkyl or a sulfur protecting group; n is 0, 1, 2, or 3, as valencypermits; and each of R^(na), R^(nb), and R^(nd) is independentlyhydrogen, an optionally substituted —C₁-C₆ alkyl, or a nitrogenprotecting group.
 13. The compound according to any one of claims 1-12or a pharmaceutically acceptable salt thereof, wherein the 5- or6-membered monocyclic heteroaryl represented by Q is selected from thefollowing:


14. The compound according to any one of claims 1-13 or apharmaceutically acceptable salt thereof, wherein the 5- or 6-memberedmonocyclic heteroaryl represented by Q is selected from the following:


15. The compound according to any one of claims 1-14 or apharmaceutically acceptable salt thereof, wherein the 5- or 6-memberedmonocyclic heteroaryl represented by Q is


16. The compound according to any one of claims 1-14 or apharmaceutically acceptable salt thereof, wherein the 5- or 6-memberedmonocyclic heteroaryl represented by Q is


17. The compound according to any one of claims 12-15 or apharmaceutically acceptable salt thereof, wherein R^(na) is hydrogen or—C₁₋₄ alkyl.
 18. The compound according to any one of claims 12-17 or apharmaceutically acceptable salt thereof, wherein each instance of R^(n)is independently hydrogen, halogen, optionally substituted C₁₋₄ alkyl,—CN, —NO₂, —N₃, —OR^(o4), —N(R^(n2))₂, —C(═O)N(R^(n2))₂, —C(═O)R^(c3),or —C(═O)OR^(o4).
 19. The compound according to any one of claims 1-18or a pharmaceutically acceptable salt thereof, wherein R¹ is hydrogen ora —C₁-C₄ alkyl.
 20. The compound according to any one of claims 1-19 ora pharmaceutically acceptable salt thereof, wherein R¹ is methyl. 21.The compound according to any one of claims 1-20 or a pharmaceuticallyacceptable salt thereof, wherein R^(j) and R^(k) are each independentlyhydrogen, a halogen, —OR^(o7), or a —C₁-C₄ alkyl, or R^(j) and R^(k) arejoined together to form ═O.
 22. The compound according to any one ofclaims 1-21 or a pharmaceutically acceptable salt thereof, wherein R^(j)and R^(k) are each hydrogen.
 23. The compound according to any one ofclaims 1-22 or a pharmaceutically acceptable salt thereof, wherein R^(a)and R^(b) are each hydrogen.
 24. The compound according to any one ofclaims 5-23, wherein q is 0 or
 1. 25. The compound according to any oneof claims 12-24, wherein n is 0 or
 1. 26. A pharmaceutical compositioncomprising an effective amount of the compound according to any one ofclaims 1-25 or a pharmaceutically acceptable salt thereof, and apharmaceutically acceptable carrier.
 27. A method for increasing thelifetime of red blood cells (RBCs) comprising contacting the red bloodcells with an effective amount of (1) the compound according to any oneof claims 1-25 or a pharmaceutically acceptable salt thereof; or (2) thepharmaceutical composition according to claim
 26. 28. The method ofclaim 27, wherein the compound or the pharmaceutical composition isadded directly to whole blood comprising the red blood cells or packedred blood cells comprising the red blood cells extracorporeally.
 29. Themethod of claim 28, wherein the compound or the pharmaceuticalcomposition is administered to a subject comprising the red blood cells.30. A method for regulating 2,3-diphosphoglycerate levels in bloodcomprising contacting the blood with an effective amount of (1) thecompound according to any one of claims 1-25 or a pharmaceuticallyacceptable salt thereof; or (2) the pharmaceutical composition accordingto claim
 26. 31. A method for treating anemia in a subject comprisingadministering to the subject an effective amount of (1) the compoundaccording to any one of claims 1-25 or a pharmaceutically acceptablesalt thereof; or (2) the pharmaceutical composition according to claim26.
 32. The method of claim 31, wherein the anemia is dyserythropoieticanemia.
 33. A method for treating hemolytic anemia in a subjectcomprising administering to the subject an effective amount of (1) thecompound according to any one of claims 1-25 or a pharmaceuticallyacceptable salt thereof; or (2) the pharmaceutical composition accordingto claim
 26. 34. The method of claim 33, wherein the hemolytic anemia ishereditary and/or congenital hemolytic anemia, acquired hemolyticanemia, or anemia as part of a multi-system disease.
 35. A method fortreating sickle cell disease in a subject comprising administering tothe subject an effective amount of (1) the compound according to any oneof claims 1-25 or a pharmaceutically acceptable salt thereof; or (2) thepharmaceutical composition according to claim
 26. 36. A method oftreating pyruvate kinase deficiency (PKD) in a subject comprisingadministering to the subject an effective amount of (1) the compoundaccording to any one of claims 1-25 or a pharmaceutically acceptablesalt thereof; or (2) the pharmaceutical composition according to claim26.
 37. A method of treating thalassemia, hereditary spherocytosis,hereditary elliptocytosis, abetalipoproteinemia or Bassen-Kornzweigsyndrome, sickle cell disease, paroxysmal nocturnal hemoglobinuria,acquired hemolytic anemia, or anemia of chronic diseases in a subjectcomprising administering to the subject an effective amount of (1) thecompound according to any one of claims 1-25 or a pharmaceuticallyacceptable salt thereof; or (2) the pharmaceutical composition accordingto claim
 26. 38. A method of treating thalassemia in a subjectcomprising administering to the subject an effective amount of (1) thecompound according to any one of claims 1-25 or a pharmaceuticallyacceptable salt thereof; or (2) the pharmaceutical composition accordingto claim
 26. 39. The method of claim 38, wherein the thalassemia isbeta-thalassemia.
 40. A method for activating mutant pyruvate kinase R(PKR) in red blood cells in a subject in need thereof comprisingadministering to the subject an effective amount of (1) the compoundaccording to any one of claims 1-25 or a pharmaceutically acceptablesalt thereof; or (2) the pharmaceutical composition according to claim26.
 41. A method for activating wild-type pyruvate kinase R (PKR) in redblood cells in a subject in need thereof comprising administering to thesubject an effective amount of (1) the compound according to any one ofclaims 1-25 or a pharmaceutically acceptable salt thereof; or (2) thepharmaceutical composition according to claim
 26. 42. A method ofincreasing amount of hemoglobin in a subject in need thereof comprisingadministering to the subject an effective amount of (1) the compoundaccording to any one of claims 1-25 or a pharmaceutically acceptablesalt thereof; or (2) the pharmaceutical composition according to claim26.